DNA-PK inhibitors

ABSTRACT

The invention relates to the use of compounds of formula (I) and isomers, salts, solvates, chemically protected forms, and prodrugs thereof, in the preparation of a medicament for inhibiting the activity of DNA-PK, wherein R 1  and R 2  are independently hydrogen, an optionally substituted C 1-7  alkyl group, C 3-20  heterocyclyl group, or C 5-20  aryl group, or may together form, along with the nitrogen atom to which they are attached, an optionally substituted heterocyclic ring having from 4 to 8 ring atoms; X and Y are selected from CR 4  and O, O and CR′ 4  and NR″ 4  and N, where the unsaturation is in the appropriate place in the ring, and where one of R 3  and R 4  or R′ 4  is an optionally substituted C 3-20  heteroaryl or C 5-20  aryl group, and the other of R 3  and R 4  or R′ 4  is H, or R 3  and R 4  or R″ 4  together are —A—B—, which collectively represent a fused optionally substituted aromatic ring. The compounds also selectively inhibit the activity of DNA-PK compared to PI 3-kinase and/or ATM.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/486,816, filed Feb. 13, 2004, now U.S. Pat. No. 7,226,918, which is anational stage filing under 35 U.S.C. §371 of PCT/GB02/03781, filed Aug.14, 2002, which claims foreign priority benefits of United KingdomApplication No. 0119865.4, filed Aug. 14, 2001. These are incorporatedherein by reference in their entirety.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

The subject matter disclosed in this patent application was developedunder a joint research agreement between Kudos Pharmaceuticals Ltd. andCancer Research Technology Ltd., formerly known as Cancer ResearchCampaign Technology Ltd.

The present invention relates to compounds which act as DNA-PKinhibitors, their use and synthesis.

The DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonineprotein kinase that is activated upon association with DNA. Biochemicaland genetic data have revealed this kinase to be composed of a largecatalytic subunit, termed DNA-PKcs, and a regulatory component termedKu. DNA-PK has been shown to be a crucial component of both the DNAdouble-strand break (DSB) repair machinery and the V(D)J recombinationapparatus. In addition, recent work has implicated DNA-PK components ina variety of other processes, including the modulation of chromatinstructure and telomere maintenance (Smith, G. C. M. and Jackson, S. P.,Genes and Dev. 13: 916-934 (1999)).

Human DNA is constantly under attack from reactive oxygen intermediatesprincipally from by-products of the oxidative metabolism we have evolvedfor energy supply. Reactive oxygen species are capable of producing DNAsingle-strand breaks and, where two of these are generated in closeproximity, DNA double strand breaks (DSBs). In addition, single- anddouble-strand breaks can be induced when a DNA replication forkencounters a damaged template, and are generated by exogenous agentssuch as ionising radiation (IR) and certain anti-cancer drugs (e.g.bleomycin). DSBs also occur as intermediates in site-specific V(D)Jrecombination, a process that is critical for the generation of afunctional vertebrate immune system. If DNA DSBs are left unrepaired orare repaired inaccurately, mutations and/or chromosomal aberrations areinduced, which in turn may lead to cell death. To combat the seriousthreats posed by DNA DSBs, eukaryotic cells have evolved severalmechanisms to mediate their repair. In higher eukaryotes, thepredominant of these mechanisms is DNA non-homologous end-joining(NHEJ), also known as illegitimate recombination. DNA-PK plays a keyrole in this pathway.

Biochemical studies on DNA-PK revealed that it is activated mostpotently by DNA DSBs, suggesting that it might play a role inrecognising DNA damage. This stimulated investigations into thepotential role of DNA-PKcs and Ku in DNA repair and led to theidentification of cell lines which are radiosensitive due to mutationsin DNA-PK components (Smith and Jackson, 1999). Cloning of the DNA-PKcscDNA revealed that it corresponds to a ˜470 kDa polypeptide, theN-terminal ˜3500 amino acid residues of which does not appear to havesignificant homology to other characterised proteins (Hartley, K. O., etal., Cell 82: 849-856 (1995)). More significantly, the C-terminal ˜500amino acid residues of DNA-PKcs comprises a catalytic domain that fallsinto the PI 3-kinase family. Although this initially suggested thatDNA-PK might be capable of phosphorylating inositol phospho-lipids, likecertain well-characterised members of the PI 3-kinase family (Toker, A.and Cantley, L. C., Nature 387: 673-676 (1997)), the available evidenceindicates that DNA-PK has protein but not lipid kinase activity (Hartleyet al. 1995; Smith et al., 1999). At a similar time to the cloning ofthe DNA-PKcs cDNA, the genes and cDNAs for a range of other large PI3-kinase like (PIKL) proteins were identified and cloned (Jackson, S.P., Cancer Surv. 28: 261-279 (1996)). These proteins have been shown tobe involved in controlling transcription, the cell-cycle and/or genomestability in organisms from yeast to man. DNA-PKcs appears to berestricted to higher eukaryotes.

Besides DNA-PKcs, probably the best characterised member of the PIKLfamily is ATM, the protein deficient in the human neurodegenerative andcancer predisposition condition ataxia-telangiectasia (A-T; Lavin, M. F.and Shiloh, Y., Annu. Rev. Immunol. 15: 177-202 (1997)). ATM has beenlinked intimately to the detection and signalling of DNA damage.

It also has been previously found that the PI 3-kinase inhibitorLY294002:

is able to inhibit DNA-PK function in vitro (Izzard, R. A., et al.,Cancer Res. 59: 2581-2586 (1999)). The IC₅₀ (concentration at which 50%of enzyme activity is lost) for LY294002 towards DNA-PK is, at ˜1 μM,the same as that for PI 3-kinase. Furthermore it has been shown thatLY294002 is also able to weakly sensitise cells to the effects of IR(Rosenzweig, K. E., et al., Clin. Cancer Res. 3: 1149-1156 (1999)).

Given the involvement of DNA-PK in DNA repair processes, and thatLY294002 has been shown to radiosensitise mammalian cells in culture, anapplication of (specific) DNA-PK inhibitory drugs would be to act asagents that will enhance the efficacy of both cancer chemotherapy andradiotherapy. DNA-PK inhibitors may also prove useful in the treatmentof retroviral mediated diseases. For example it has been demonstratedthat loss of DNA-PK activity severely represses the process ofretroviral integration (Daniel R, et al., Science, 284:644-7 (1999)).DNA-PK inhibitors may also have potential as modulators of the immunesystem. DNA-PK has also been shown to play an important role in telomeremaintenance, and hence inhibitors of DNA-PK may play a role inmodulating telomere functions (Goytisolo, et al, Mol. Cell. Biol.,21:3642-3651 (2001).

The present inventors have now discovered compounds which exhibitinhibition of DNA-PK; these compounds also exhibit selective inhibitionof DNA-PK over the PI 3-kinase family members PI 3-kinase and ATM.

Accordingly, the first aspect of the invention provides for the use ofcompounds of formula I:

and isomers, salts, solvates, chemically protected forms, and prodrugsthereof, in the preparation of a medicament for inhibiting the activityof DNA-PK, wherein:R¹ and R² are independently hydrogen, an optionally substituted C₁₋₇alkyl group, C₃₋₂₀ heterocyclyl group, or C₅₋₂₀ aryl group, or maytogether form, along with the nitrogen atom to which they are attached,an optionally substituted heterocyclic ring having from 4 to 8 ringatoms;X and Y are selected from CR⁴ and O, O and CR′⁴ and NR′⁴ and N, wherethe unsaturation is in the appropriate place in the ring, and where oneof R³ and R⁴ or R′⁴ is an optionally substituted C₃₋₂₀ heteroaryl orC₅₋₂₀ aryl group, and the other of R³ and R⁴ or R′⁴ is H, or R³ and R⁴or R″⁴ together are —A—B—, which collectively represent a fusedoptionally substituted aromatic ring;except that when X and Y are CR⁴ and O, R³ and R⁴ together form a fusedbenzene ring, and R¹ and R² together with the N to which they areattached form a morpholino group, then the fused benzene does not bearas a sole substituent a phenyl substituent at the 8-position.

Thus, the three different possibilities for X and Y results in compoundsof formulae Ia, Ib and Ic:

One aspect of the first aspect of the present invention relates tocompounds of formulae Ia or Ib, where one R³ and R⁴ (or R′⁴) is a C₃₋₂₀heteroaryl or C₅₋₂₀ aryl group, and the other of R³ and R⁴ (or R′⁴) isH.

Another aspect of the first aspect of the present invention relates tocompounds of formulae Ia and Ic, where R³ and R⁴ or R″⁴ together are—A—B—, which collectively represent a fused optionally substitutedaromatic ring, with the proviso given above.

It is preferred that the medicament of the first aspect selectivityinhibits the activity of DNA-PK compared to PI 3-kinase and/or ATM.Selectivity is an important issue as inhibition of other PI 3-kinasefamily members may lead to unwanted side-effects associated with theloss of function of those enzymes.

A second aspect of the invention provides for the use of compounds asdefined in the first aspect of the invention in the preparation of amedicament for use as an adjunct in cancer therapy or for potentiatingtumour cells for treatment with ionising radiation or chemotherapeuticagents.

A third aspect of the invention provides for the use of compounds in thepreparation of a medicament for the treatment of retroviral mediateddiseases or disease ameliorated by the inhibition of DNA-PK.

A further aspect of the invention provides an active compound asdescribed herein for use in a method of treatment of the human or animalbody, preferably in the form of a pharmaceutical composition.

Another aspect of the invention provides a method of inhibiting DNA-PKin vitro or in vivo, comprising contacting a cell with an effectiveamount of an active compound as described herein.

A further aspect of the present invention provides novel compounds asdescribed herein.

DEFINITIONS

The term “aromatic ring” is used herein in the conventional sense torefer to cyclic aromatic rings, that is, cyclic structures having 5 to 7atoms in a ring with delocalised n-electron orbitals. Preferably,aromatic rings are those which meet Hückel's 4n+2 rule, ie. where thenumber of n-electrons is 4n+2, n representing the number of ring atoms.It is preferred that the aromatic ring has six atoms. In such a case, itis further preferred that the four atoms additional to the core moietythat make up the aromatic ring are all carbon, which yields compounds ofthe following general structure:

wherein X′ and Y′ are either C and O or N and N, respectively; andwhere R⁵, R⁶, R⁷, and R⁸ are preferably independently selected fromhydrogen, C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, hydroxy, C₁₋₇alkoxy (including C₁₋₇ alkyl-C₁₋₇ alkoxy and C₃₋₂₀ aryl-C₁₋₇ alkoxy) andacyloxy or adjacent pairs of substituents (i.e. R⁵ and R⁶, R⁶ and R⁷, R⁷and R⁸) form, together with the atoms to which they are attached, anoptionally substituted aromatic or carbocyclic ring.

The fused aromatic ring represented by —A—B— may be substituted by oneor more of the following groups: C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀aryl, hydroxy, C₁₋₇ alkoxy (including C₁₋₇ alkyl-C₁₋₇ alkoxy and C₃₋₂₀aryl-C₁₋₇ alkoxy) and acyloxy; adjacent pairs of substituents may form,together with the atoms to which they are attached, an optionallysubstituted aromatic or carbocyclic ring.

The term carbocyclic ring refers to a ring formed from 5 to 7 covalentlylinked carbon atoms. The ring may contain one or more carbon-carbondouble bonds. Examples of carbocyclic rings include cyclopentane,cyclohexane, cycloheptane, cyclopentene, cyclohexene and cycloheptene.

C₁₋₇ alkyl: The term “C₁₋₇ alkyl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a C₁₋₇hydrocarbon compound having from 1 to 7 carbon atoms, which may bealiphatic or alicyclic, or a combination thereof, and which may besaturated, partially unsaturated, or fully unsaturated.

Examples of saturated linear C₁₋₇ alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl (amyl).

Examples of saturated branched C₁₋₇ alkyl groups include, but are notlimited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl, andneo-pentyl.

Examples of saturated alicyclic C₁₋₇ alkyl groups (also referred to as“C₃₋₇ cycloalkyl” groups) include, but are not limited to, groups suchas cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well assubstituted groups (e.g., groups which comprise such groups), such asmethylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl,dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl,methylcyclohexyl, dimethylcyclohexyl, cyclopropylmethyl andcyclohexylmethyl.

Examples of unsaturated C₁₋₇ alkyl groups which have one or morecarbon-carbon double bonds (also referred to as “C₂₋₇alkenyl” groups)include, but are not limited to, ethenyl (vinyl, —CH═CH₂), 2-propenyl(allyl, —CH—CH═CH₂), isopropenyl (—C(CH₃)═CH₂), butenyl, pentenyl, andhexenyl.

Examples of unsaturated C₁₋₇ alkyl groups which have one or morecarbon-carbon triple bonds (also referred to as “C₂₋₇ alkynyl” groups)include, but are not limited to, ethynyl (ethinyl) and 2-propynyl(propargyl).

Examples of unsaturated alicyclic (carbocyclic) C₁₋₇ alkyl groups whichhave one or more carbon-carbon double bonds (also referred to as“C₃₋₇cycloalkenyl” groups) include, but are not limited to,unsubstituted groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl,and cyclohexenyl, as well as substituted groups (e.g., groups whichcomprise such groups) such as cyclopropenylmethyl andcyclohexenylmethyl.

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl”, as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a C₃₋₂₀ heterocyclic compound, said compound havingone ring, or two or more rings (e.g., spiro, fused, bridged), and havingfrom 3 to 20 ring atoms, atoms, of which from 1 to 10 are ringheteroatoms, and wherein at least one of said ring(s) is a heterocyclicring. Preferably, each ring has from 3 to 7 ring atoms, of which from 1to 4 are ring heteroatoms. “C₃₋₂₀” denotes ring atoms, whether carbonatoms or heteroatoms.

Examples of C₃₋₂₀ heterocyclyl groups having one nitrogen ring atominclude, but are not limited to, those derived from aziridine,azetidine, pyrrolidines (tetrahydropyrrole), pyrroline (e.g.,3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole (isopyrrole,isoazole), piperidine, dihydropyridine, tetrahydropyridine, and azepine.

Examples of C₃₋₂₀ heterocyclyl groups having one oxygen ring atominclude, but are not limited to, those derived from oxirane, oxetane,oxolane (tetrahydrofuran), oxole (dihydrofuran), oxane(tetrahydropyran), dihydropyran, pyran (C₆), and oxepin. Examples ofsubstituted C₃₋₂₀ heterocyclyl groups include sugars, in cyclic form,for example, furanoses and pyranoses, including, for example, ribose,lyxose, xylose, galactose, sucrose, fructose, and arabinose.

Examples of C₃₋₂₀ heterocyclyl groups having one sulphur ring atominclude, but are not limited to, those derived from thiirane, thietane,thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), andthiepane.

Examples of C₃₋₂₀ heterocyclyl groups having two oxygen ring atomsinclude, but are not limited to, those derived from dioxolane, dioxane,and dioxepane.

Examples of C₃₋₂₀ heterocyclyl groups having two nitrogen ring atomsinclude, but are not limited to, those derived from imidazolidine,pyrazolidine (diazolidine), imidazoline, pyrazoline (dihydropyrazole),and piperazine.

Examples of C₃₋₂₀ heterocyclyl groups having one nitrogen ring atom andone oxygen ring atom include, but are not limited to, those derived fromtetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole,dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, andoxazine.

Examples of C₃₋₂₀ heterocyclyl groups having one oxygen ring atom andone sulphur ring atom include, but are not limited to, those derivedfrom oxathiolane and oxathiane (thioxane).

Examples of C₃₋₂₀ heterocyclyl groups having one nitrogen ring atom andone sulphur ring atom include, but are not limited to, those derivedfrom thiazoline, thiazolidine, and thiomorpholine.

Other examples of C₃₋₂₀ heterocyclyl groups include, but are not limitedto, oxadiazine and oxathiazine.

Examples of heterocyclyl groups which additionally bear one or more oxo(═O) groups, include, but are not limited to, those derived from:

-   -   C₅ heterocyclics, such as furanone, pyrone, pyrrolidone        (pyrrolidinone), pyrazolone (pyrazolinone), imidazolidone,        thiazolone, and isothiazolone;    -   C₆ heterocyclics, such as piperidinone (piperidone),        piperidinedione, piperazinone, piperazinedione, pyridazinone,        and pyrimidinone (e.g., cytosine, thymine, uracil), and        barbituric acid;    -   fused heterocyclics, such as oxindole, purinone (e.g., guanine),        benzoxazolinone, benzopyrone (e.g., coumarin);    -   cyclic anhydrides (—C(═O)—O—C(═O)— in a ring), including but not        limited to maleic anhydride, succinic anhydride, and glutaric        anhydride;    -   cyclic carbonates (—O—C(═O)—O— in a ring), such as ethylene        carbonate and 1,2-propylene carbonate;    -   imides (—C(═O)—NR—C(═O)— in a ring), including but not limited        to, succinimide, maleimide, phthalimide, and glutarimide;    -   lactones (cyclic esters, —O—C(═O)— in a ring), including, but        not limited to, β-propiolactone, γ-butyrolactone,        δ-valerolactone (2-piperidone), and ε-caprolactone;    -   lactams (cyclic amides, —NR—C(═O)— in a ring), including, but        not limited to, β-propiolactam, γ-butyrolactam (2-pyrrolidone),        δ-valerolactam, and ε-caprolactam;    -   cyclic carbamates (—O—C(═O)—NR— in a ring), such as        2-oxazolidone;    -   cyclic ureas (—NR—C(═O)—NR— in a ring), such as 2-imidazolidone        and pyrimidine-2,4-dione (e.g., thymine, uracil).

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of a C₅₋₂₀ aromatic compound, said compound having one ring,or two or more rings (e.g., fused), and having from 5 to 20 ring atoms,and wherein at least one of said ring(s) is an aromatic ring.Preferably, each ring has from 5 to 7 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”, inwhich case the group may conveniently be referred to as a “C₅₋₂₀carboaryl” group.

Examples of C₅₋₂₀ aryl groups which do not have ring heteroatoms (i.e.C₅₋₂₀ carboaryl groups) include, but are not limited to, those derivedfrom benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), anthracene (C₁₄),phenanthrene (C₁₄), naphthacene (C₁₈) and pyrene (C₁₆).

Examples of aryl groups which comprise fused rings, one of which is notan aromatic ring, include, but are not limited to, groups derived fromindene and fluorene.

Alternatively, the ring atoms may include one or more heteroatoms,including but not limited to oxygen, nitrogen, and sulphur, as in“heteroaryl groups”. In this case, the group may conveniently bereferred to as a “C₅₋₂₀ heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteroatoms. Preferably, each ring hasfrom 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

Examples of C₅₋₂₀ heteroaryl groups include, but are not limited to, C₅heteroaryl groups derived from furan (oxole), thiophene (thiole),pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole),triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, andoxatriazole; and C₆ heteroaryl groups derived from isoxazine, pyridine(azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine), triazine, tetrazole,and oxadiazole (furazan).

Examples of C₅₋₂₀ heterocyclic groups (some of which are C₅₋₂₀heteroaryl groups) which comprise fused rings, include, but are notlimited to, C₉ heterocyclic groups derived from benzofuran,isobenzofuran, indole, isoindole, purine (e.g., adenine, guanine),benzothiophene, benzimidazole; C₁₀ heterocyclic groups derived fromquinoline, isoquinoline, benzodiazine, pyridopyridine, quinoxaline; C₁₃heterocyclic groups derived from carbazole, dibenzothiophene,dibenzofuran; C₁₄ heterocyclic groups derived from acridine, xanthene,phenoxathiin, phenazine, phenoxazine, phenothiazine.

The above C₁₋₇ alkyl, C₃₋₂₀ heterocyclyl, and C₅₋₂₀ aryl groups, whetheralone or part of another substituent, may themselves optionally besubstituted with one or more groups selected from themselves and theadditional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇ alkyl group.

C₁₋₇ alkoxy: —OR, wherein R is a C₁₋₇ alkyl group. Examples of C₁₋₇alkoxy groups include, but are not limited to, —OCH₃ (methoxy), —OCH₂CH₃(ethoxy) and —OC(CH₃)₃ (tert-butoxy).

Oxo (keto, -one): ═O. Examples of cyclic compounds and/or groups having,as a substituent, an oxo group (═O) include, but are not limited to,carbocyclics such as cyclopentanone and cyclohexanone; heterocyclics,such as pyrone, pyrrolidone, pyrazolone, pyrazolinone, piperidone,piperidinedione, piperazinedione, and imidazolidone; cyclic anhydrides,including but not limited to maleic anhydride and succinic anhydride;cyclic carbonates, such as propylene carbonate; imides, including butnot limited to, succinimide and maleimide; lactones (cyclic esters,—O—C(═O)— in a ring), including, but not limited to, β-propiolactone,γ-butyrolactone, δ-valerolactone, and ε-caprolactone; and lactams(cyclic amides, —NH—C(═O)— in a ring), including, but not limited to,β-propiolactam, γ-butyrolactam (2-pyrrolidone), δ-valerolactam, andε-caprolactam.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl), aC₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —COOH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O) Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyland phthalimidyl:

Acylureido: —N(R¹)C(O)NR²C(O)R³ wherein R¹ and R² are independentlyureido substituents, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. R³ is an acyl group as defined for acyl groups. Examples ofacylureido groups include, but are not limited to, —NHCONHC(O)H,—NHCONMeC(O)H, —NHCONEtC(O)H, —NHCONMeC(O)Me, —NHCONEtC(O)Et,—NMeCONHC(O)Et, —NMeCONHC(O)Me, —NMeCONHC(O)Et, —NMeCONMeC(O)Me,—NMeCONEtC(O)Et, and —NMeCONHC(O)Ph.

Carbamate: —NR¹—C(O)—OR² wherein R¹ is an amino substituent as definedfor amino groups and R² is an ester group as defined for ester groups.Examples of carbamate groups include, but are not limited to,—NH—C(O)—O-Me, —NMe-C(O)—O-Me, —NH—C(O)—O-Et, —NMe-C(O)—O-t-butyl, and—NH—C(O)—O-Ph.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Examples of amino groups include, but are not limited to,—NH₂, —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples ofcyclic amino groups include, but are not limited to, aziridino,azetidino, pyrrolidino, piperidino, piperazino, morpholino, andthiomorpholino.

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇ alkyl group.

Amidine: —C(═NR)NR₂, wherein each R is an amidine substituent, forexample, hydrogen, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. An example of anamidine group is —C(═NH)NH₂.

Carbazoyl (hydrazinocarbonyl): —C(O)—NN—R¹ wherein R¹ is an aminosubstituent as defined for amino groups. Examples of azino groupsinclude, but are not limited to, —C(O)—NN—H, —C(O)—NN-Me, —C(O)—NN-Et,—C(O)—NN-Ph, and —C(O)—NN—CH₂-Ph.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkylthiogroup), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfone groupsinclude, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl, mesyl),—S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃, —S(═O)₂C₄F₉ (nonaflyl),—S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂Ph (phenylsulfonyl),4-methylphenylsulfonyl (tosyl), 4-bromophenylsulfonyl(brosyl), and4-nitrophenyl (nosyl).

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ and —OS(═O)₂CH₂CH₃.

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groupsinclude, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃) S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Sulfamyl: —S(═O)NR¹R², wherein R¹ and R² are independently aminosubstituents, as defined for amino groups. Examples of sulfamyl groupsinclude, but are not limited to, —S(═O)NH₂, —S(═O)NH(CH₃),—S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅. A specialclass of sulfonamino groups are those derived from sultams—in thesegroups one of R¹ and R is a C₅₋₂₀ aryl group, preferably phenyl, whilstthe other of R¹ and R is a bidentate group which links to the C₅₋₂₀ arylgroup, such as a bidentate group derived from a C₁₋₇ alkyl group.Examples of such groups include, but are not limited to:

2,3-dihydro-tenzo[d]isothiazole-1,1-dioxide-2-yl

1,3-dihydro-benzo[c]isothiazole-2,2-dioxide-1-yl

3,4-dihydro-2H-benzo[e][1,2]thiazine-1,1-dioxide-2-yl

Phosphoramidite: —OP(OR¹)—NR₂₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr) 2.

Phosphoramidate: —OP(═O) (OR¹)—NR₂₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O) (OCH₂CH₃)—N(CH₃)₂,—OP(═O) (OCH₂CH₃)—N(i-Pr)₂, and —OP(═O) (OCH₂CH₂CN)—N(i-Pr)₂.

In many cases, substituents may themselves be substituted. For example,a C₁₋₇ alkoxy group may be substituted with, for example, a C₁₋₇ alkyl(also referred to as a C₁₋₇ alkyl-C₁₋₇alkoxy group), for example,cyclohexylmethoxy, a C₃₋₂₀ heterocyclyl group (also referred to as aC₅₋₂₀ aryl-C₁₋₇ alkoxy group), for example phthalimidoethoxy, or a C₅₋₂₀aryl group (also referred to as a C₅₋₂₀ aryl-C₁₋₇alkoxy group), forexample, benzyloxy.

Includes Other Forms

Included in the above are the well known ionic, salt, solvate, andprotected forms of these substituents. For example, a reference tocarboxylic acid (—COOH) also includes the anionic (carboxylate) form(—COO⁻), a salt or solvate thereof, as well as conventional protectedforms. Similarly, a reference to an amino group includes the protonatedform (—N⁺HR¹R²), a salt or solvate of the amino group, for example, ahydrochloride salt, as well as conventional protected forms of an aminogroup. Similarly, a reference to a hydroxyl group also includes theanionic form (—O⁻), a salt or solvate thereof, as well as conventionalprotected forms of a hydroxyl group.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- andexo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+)and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms;synclinal- and anticlinal-forms; α- and β-forms; axial and equatorialforms; boat-, chair-, twist-, envelope-, and halfchair-forms; andcombinations thereof, hereinafter collectively referred to as “isomers”(or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including 160 and 180; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g., fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts”, J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: acetic, propionic, succinic, glycolic, stearic, palmitic,lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic,hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic,pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric,phenylsulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethanedisulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, andgluconic. Examples of suitable polymeric anions include, but are notlimited to, those derived from the following polymeric acids: tannicacid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form.

The term “chemically protected form”, as used herein, pertains to acompound in which one or more reactive functional groups are protectedfrom undesirable chemical reactions, that is, are in the form of aprotected or protecting group (also known as a masked or masking groupor a blocked or blocking group). By protecting a reactive functionalgroup, reactions involving other unprotected reactive functional groupscan be performed, without affecting the protected group; the protectinggroup may be removed, usually in a subsequent step, withoutsubstantially affecting the remainder of the molecule. See, for example,Protective Groups in Organic Synthesis (T. Green and P. Wuts, Wiley,1999).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl,benzhydryl(diphenylmethyl), or trityl (triphenylmethyl)ether; atrimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester(—OC(═O)CH₃, —OAc).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (>C═O) is convertedto a diether (>C(OR)₂), by reaction with, for example, a primaryalcohol. The aldehyde or ketone group is readily regenerated byhydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amideor a urethane, for example, as: a methyl amide (—NHCO—CH₃); a benzyloxyamide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxy amide (—NHCO—OC(CH₃)₃,—NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH₃)₂C₆H₄C₆H₅,—NH-Bpoc), as a 9-fluorenylmethoxy amide (—NH-Fmoc), as a6-nitroveratryloxy amide (—NH-Nvoc), as a 2-trimethylsilylethyloxy amide(—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as anallyloxy amide (—NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide(—NH-Psec); or, in suitable cases, as an N-oxide (>NO$).

For example, a carboxylic acid group may be protected as an ester forexample, as: an C₁₋₇ alkyl ester (e.g. a methyl ester; a t-butyl ester);a C₁₋₇ haloalkyl ester (e.g., a C₁₋₇ trihaloalkyl ester); a triC₁₋₇alkylsilyl-C₁₋₇ alkyl ester; or a C₅₋₂₀ aryl-C₁₋₇ alkyl ester (e.g. abenzyl ester; a nitrobenzyl ester); or as an amide, for example, as amethyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; an acetamidomethyl ether(—S—CH₂NHC(═O)CH₃).

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in the form of a prodrug. The term “prodrug”, as usedherein, pertains to a compound which, when metabolised (e.g. in vivo),yields the desired active compound. Typically, the prodrug is inactive,or less active than the active compound, but may provide advantageoushandling, administration, or metabolic properties.

For example, some prodrugs are esters of the active compound (e.g. aphysiologically acceptable metabolically labile ester). Duringmetabolism, the ester group (—C(═O)OR) is cleaved to yield the activedrug. Such esters may be formed by esterification, for example, of anyof the carboxylic acid groups (—C(═O)OH) in the parent compound, with,where appropriate, prior protection of any other reactive groups presentin the parent compound, followed by deprotection if required. Examplesof such metabolically labile esters include those wherein R is C₁₋₇alkyl (e.g. -Me, -Et); C₁₋₇ aminoalkyl (e.g. aminoethyl;2-(N,N-diethylamino)ethyl; 2-(4-morpholino) ethyl); and acyloxy-C₁₋₇alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl;acetoxymethyl; 1-acetoxyethyl;1-(1-methoxy-1-methyl)ethyl-carbonxyloxyethyl; 1-(benzoyloxy)ethyl;isopropoxy-carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl;cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl;cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl;(4-tetrahydropyranyloxy) carbonyloxymethyl;1-(4-tetrahydropyranyloxy)carbonyloxyethyl;(4-tetrahydropyranyl)carbonyloxymethyl; and1-(4-tetrahydropyranyl)carbonyloxyethyl).

Also, some prodrugs are activated enzymatically to yield the activecompound, or a compound which, upon further chemical reaction, yieldsthe active compound. For example, the prodrug may be a sugar derivativeor other glycoside conjugate, or may be an amino acid ester derivative.

Selective Inhibition

‘Selective inhibition’ means the inhibition of one enzyme to a greaterextent than the inhibition of one or more other enzymes. Thisselectivity is measurable by comparing the concentration of a compoundrequired to inhibit 50% of the activity (IC₅₀) of one enzyme against theconcentration of the same compound required to inhibit 50% of theactivity (IC₅₀) of the other enzyme (see below). The result is expressedas a ratio. If the ratio is greater than 1, then the compound testedexhibits some selectivity in its inhibitory action.

The compounds of the present invention preferably exhibit a selectivityof greater than 3, 10, 20 or 50 against DNA-PK over PI 3-kinase.

The compounds of the present invention preferably exhibit a selectivityof greater than 5, 10, 50 or 100 against DNA-PK over ATM.

It is preferred that the IC₅₀s used to determine selectivity aredetermined using the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of preferred compounds of formula Ib.

FIG. 2 shows the structure of preferred compounds of formula Ic.

FIG. 3 shows the structure of preferred compounds of formula Ia.

FIG. 4 shows the structures of further preferred compounds of formulaIa.

FIG. 5 shows the structures of further preferred compounds of formulaIa.

FURTHER PREFERENCES

In formula I, when R¹ and R² form, along with the nitrogen atom to whichthey are attached, a heterocyclic ring having from 4 to 8 atoms, thismay form part of a C₄₋₂₀ heterocyclyl group defined above (except with aminimum of 4 ring atoms), which must contain at least one nitrogen ringatom. It is preferred that R¹ and R² form, along with the nitrogen atomto which they are attached, a heterocyclic ring having 5, 6 or 7 atoms,more preferably 6 ring atoms.

Single rings having one nitrogen atom include azetidine, azetidine,pyrrolidine (tetrahydropyrrole), pyrroline (e.g., 3-pyrroline,2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole),piperidine, dihydropyridine, tetrahydropyridine, and azepine; twonitrogen atoms include imidazolidine, pyrazolidine (diazolidine),imidazoline, pyrazoline (dihydropyrazole), and piperazine; one nitrogenand one oxygen include tetrahydrooxazole, dihydrooxazole,tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine,dihydrooxazine, and oxazine; one nitrogen and one sulphur includethiazoline, thiazolidine, and thiomorpholine.

Preferred rings are those containing one heteroatom in addition to thenitrogen, and in particular, the preferred heteroatoms are oxygen andsulphur. Thus preferred groups include morpholino, thiomorpholino,thiazolinyl. Preferred groups without a further heteroatom includepyrrolidino.

The most preferred groups are morpholino and thiomorpholino.

As mentioned above, these heterocyclic groups may themselves besubstituted; a preferred class of substituent is a C₁₋₇ alkyl group.When the heterocyclic group is morpholino, the substituent group orgroups are preferably methyl or ethyl, and more preferably methyl. Asole methyl substituent is most preferably in the 2 position.

As well as the single ring groups listed above, rings with bridges orcross-links are also envisaged. Examples of these types of ring wherethe group contains a nitrogen and an oxygen atom are:

These are named 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl,6-oxa-3-aza-bicyclo[3.1.0]hex-3-yl, 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl,and 7-oxa-3-aza-bicyclo[4.1.0]hept-3-yl, respectively.

The proviso as set in the first aspect of the invention preferablyexcludes compounds where X and Y are CR⁴ and O, R³ and R⁴ together forma fused benzene ring, and R¹ and R² together with the N to which theyare attached form a morpholino group, and the fused benzene does notbear as a sole substituent a substituent at the 8-position. Analternative preferred embodiment is to exclude compounds where X and Yare CR⁴ and O, R³ and R⁴ together form a fused benzene ring, and R¹ andR² together with the N to which they are attached form a morpholinogroup, and the fused benzene does not bear a sole substituent that is aphenyl group.

Preferred Aspects of Compounds of Formula Ia

It is preferred that R¹ and R² in formula Ia together form a morpholinogroup.

In one preferred aspect of compounds of formula Ia, R⁴ is preferably H.R³ is preferably a C₅₋₂₀ aryl group, more preferably a C₅₋₂₀ carboarylgroup, and in particular an optionally substituted phenyl group.Preferred substituents include halo (particularly fluoro and chloro),C₁₋₇ alkyl (particularly C₁, alkyl or t-butyl), ether, alkoxy (inparticular methoxy), nitro, cyano, acyl, formyl, ester, acyloxy,hydroxy, carboxy, C₅₋₂₀ aryl (particularly phenyl), C₃₋₂₀ heterocyclyl,acylamido, acylureido, thioureido, carbamate, carbazoyl, amido, andamino.

When R³ is C₅₋₂₀ aryl, examples of preferred groups include optionallysubstituted napthalene, quiniline, pyridine, indole, indazole, pyrazine,pyrrole, imidazole, thiophene, thiazole, benzo[b]thiophene, furan andbenzofuran.

R³ may be substituted with one or more substituents, preferably onesubstituent. Preferably R³ is a mono substituted phenyl.

Where R³ is a C₅₋₂₀ aryl group other than phenyl, preferred substituentsinclude C₁₋₇ alkyl, formyl and ether (in particular alkoxy).

When R³ is a C₃₋₂₀ aryl group, the substituents may be at any positionon the aryl group. Accordingly, when R³ is an optionally substitutedphenyl the substituents may be at the ortho-(2-), meta-(3-) or para-(4-)position. It is generally preferred that the substituents are in thepara- (or 4-) position. Preferably R³ is a 4-substituted phenyl. Thenature of the substituent is discussed below.

Preferred R³ Substituents

A first group of preferred substituents include halo (particularlyfluoro and chloro), C₁₋₇ alkyl (particularly t-butyl) and alkoxy(particularly methoxy).

Preferred compounds of this type include2-(morpholin-4-yl)-6-phenyl-pyran-4-one (Compound 285),2-(4-chlorophenyl)-6-(morpholin-4-yl)-pyran-4-one (Compound 284),2-(3-methoxyphenyl)-6-(morpholin-4-yl)-pyran-4-one (Compound 287),2-(4-tert-butyl-phenyl)-6-(morpholin-4-yl)-pyran-4-one (Compound 289),2-(2-methoxyphenyl)-6-(morpholin-4-yl)-pyran-4-one (Compound 286),2-(4-Methoxyphenyl)-6-(morpholin-4-yl)-pyran-4-one (Compound 288),6-(4-fluorophenyl)-2-(morpholin-4-yl)pyran-4-one (Compound 292),6-(3-fluorophenyl)-2-(morpholin-4-yl)pyran-4-one (Compound 291) and6-(2-fluorophenyl)-2-(morpholin-4-yl)pyran-4-one (Compound 290), with6-(4-fluorophenyl)-2-(morpholin-4-yl)pyran-4-one (Compound 292) beingthe most preferred. (See FIG. 3).

Preferably the substituent is C₁₋₇ alkyl, and in particular C₁ alkyl ort-butyl. Preferably R³ is substituted C₁₋₇ alkyl (i.e. C₁₋₇ alkylene),and preferred substituents are discussed below.

A second group of preferred substituents include acylamido, acylureido,thioureido, carbamate, carbazoyl, amido and amino.

In accordance with the definitions above, it is preferred that theamino, acyl, ester, acyloxy and amide groups of the preferred acylamido,acylureido, thioureido, carbamate, carbazoyl, amido and aminosubstituents are independently H, C₁₋₇ alkyl (including substituted C₁₋₇alkyl, i.e. C₁₋₇ alkylene), C₅₋₂₀ aryl (including C₅₋₂₀ aralkyl), C₃₋₂₀heterocycle or two of the groups form a heterocycle. Preferably theamino, acyl, ester, acyloxy and amide groups are independently H, C₁alkyl, phenyl or heterocyclyl containing 3 to 7 ring atoms, or two ormore groups form a heterocyclyl ring.

Where the amino, acyl, ester, acyloxy and amide groups of the secondgroup of preferred substituents are C₅₋₂₀ aryl it is preferred that theC₅₋₂₀ aryl is phenyl, benzyl, pyridine, pyrimidine, oxazine, furan,thiophene, imidazole or oxazole. Where the amino, acyl, ester, acyloxyand amide groups of the second group of preferred substituents are C₃₋₂₀heterocyclyl they preferably have 3 to 7 ring atoms and preferablycontain from 1 to 4 ring heteroatoms.

Where two of the amino, acyl, ester, acyloxy and amide groups of thesecond group of preferred substituents form a heterocyclyl comprising aheteroatom from the preferred substituent, the heterocyclyl preferablycomprises 3 to 7 ring members. Preferably the heterocyclyl contains from1 to 4 ring heteroatoms. Examples of preferred heterocyclyls includethose derived from piperazine and azepine, morpholine andthiomorpholine.

Further Substitution

In general, where R³ of formula Ia is C₅₋₂₀ aryl group or C₅₋₂₀carboaryl group, it is preferred that the C₅₋₂₀ aryl or C₅₋₂₀ carboarylgroup is substituted. It is also preferred that when R³ is optionallysubstituted phenyl, the optionally substituted phenyl group is itselffurther substituted. It is particularly preferred that the preferred R³substituents discussed above are further substituted (i.e. the C₁₋₇alkyl, ether, alkoxy, acyl, ester, acyloxy, C₅₋₂₀ aryl, C₃₋₂₀heterocyclyl, acylamido, acylureido, thioureido, carbamate, carbazoyl,amido, and amino are themselves further substituted). The furthersubstitution may comprise any of the substituents or groups describedherein but is preferably one or more of halo (in particular fluoro orchloro), nitro, cyano (in particular methyl- or ethylcyano), hydroxy,ester, ether, alkoxy (in particular methoxy), acyloxy, acyl, thioether,carboxy, amino (in particular —NH₂ and —NMe₂), C₅₋₂₀ aryl (in particularphenyl, thiophene and furan), thioether, carbamate, C₁₋₇ alkyl and C₃₋₂₀heterocyclyl (in particular N-, O- and S-containing heterocyclylincluding tetrahydrofuran, piperidine and pyrrolidine). Thus, forexample, R³ may be haloalkyl substituted phenyl, cyanoalkyl substitutedphenyl or trifluoromethoxy substituted phenyl.

Accordingly, in a preferred class of compounds in which R³ of formula Iais C₁₋₇ alkyl substituted phenyl it is preferred that the alkylsubstituent is further substituted (to form C₁₋₇ alkylene) by halo,amino, amido, acylamido, ester or acyloxy groups.

In the preferred class of compounds in which R³ is a phenyl substitutedwith acylamido, acylureido, thioureido, carbamate, carbazoyl, amido oramino, it is preferred that these substituents are further substituted,preferably by halo (in particular fluoro or chloro), nitro, cyano (inparticular methyl- or ethylcyano), hydroxy, ester, ether, acyloxy, acyl,thioether, carboxy, C₅₋₂₀ aryl, C₁₋₇ alkyl and C₃₋₂₀ heterocyclyl (inparticular N-, O- and S-containing heterocyclyl).

In a preferred group of compounds in this preferred aspect of compoundsof formula Ia R³ is aminomethyl substituted phenyl, where the aminogroup is preferably further substituted as stated above. Preferably theaminomethyl group is at the 3- or 4-position on the phenyl.

In another preferred group of compounds in this preferred aspect ofcompounds of formula Ia R³ is amido substituted phenyl, where the amidogroup is preferably further substituted as stated above. Preferably theamido group is at the 3- or 4-position on the phenyl.

In another preferred group of compounds in this preferred aspect ofcompounds of formula Ia R³ is acylamido substituted phenyl, where theacylamido group is preferably further substituted as stated above.Preferably the acylamido group is at the 3- or 4-position on the phenyl.

In another preferred group of compounds in this preferred aspect ofcompounds of formula Ia R³ is amino substituted phenyl, where the aminogroup is preferably further substituted as stated above. Preferably theamino group is at the 3- or 4-position on the phenyl.

In another preferred aspect of compounds of formula Ia, where R³ and R⁴together are —A—B—, which collectively represent a fused aromatic ringwhich is benzene, it is preferred that the 5 position is unsubstituted(i.e. R⁵=H) and that one or two of the 6, 7 and 8 positions aresubstituted. Preferably only one of the 6, 7 and 8 positions issubstituted. Preferably the 7-position is substituted. Preferably thesubstituents are selected from halo (in particular bromo); ether (inparticular aralkyl ethers and especially where the aryl is furthersubstituted with halo, C₁₋₇ alkyl, alkoxy or nitro); C₅₋₂₀ aryl (inparticular napth-1-yl and napth-2-yl) optionally substituted by C₁₋₇alkyl (in particular methyl) including C₁₋₇ alkyl (in particular propyl)substituted by C₅₋₂₀ aryl (preferably phenyl); C₅₋₂₀ heteroaryl (inparticular benzo[b]thiophen-3-yl, benzo[b]thiophen-2-yl, thiophen-3-yl,thiophen-2-yl, furan-2-yl, indol-6-yl, quinoline-8-yl,phenoxathiin-4-yl) optionally substituted by acyl (in particular5-acetyl-thiophen-2-yl); C₃₋₂₀ heterocyclyl; amino; sulfonoxy(especially where the sulfonoxy substitutent is haloalkyl, in particularCF₃).

In another preferred class of compounds in this preferred aspect ofcompounds of formula Ia, it is preferred that the fused benzene ring(i.e. —A—B—) is substituted at the 8-position with a C₃₋₂₀ heterocyclylgroup. Preferably the heterocyclyl group is a tricyclic structure.Preferably the group comprises oxygen and/or sulfur heteroatoms and isbased on the carbazole or anthracene system. Preferably a sulfur atomand/or oxygen atom is present in the central ring of the carbazole oranthracene systems.

In the preferred group of compounds where the 6, 7, or 8 substituent isphenyl, it is preferred that the phenyl is itself further substituted.Preferably the phenyl is mono substituted but it may also be disubstituted. Preferred substitutents include ester (especially where theester substitutent is aralkyl, in particular benzyl, or C₁₋₇ alkyl, inparticular methyl or ethyl); ether (especially where the ethersubstituent is C₁₋₇ alkyl, in particular methyl or trifluoromethyl, orarylalkyl, in particular benzyl); cyano; acyl (especially where the acylsubstituent is C₁₋₇ alkyl, in particular methyl); C₅₋₂₀ aryl (inparticular phenyl); acylamido (especially where the acyl substituent isC₁₋₇ alkyl, in particular methyl); halo (in particular chloro); C₁₋₇alkyl (preferably methyl or ethyl) especially C₁₋₇ alkyl substituted byhydroxy, fluoro, acylamido (in particular phthalimidyl) and C₁₋₇ alkylsubstituted with an ester with the ester substituent being C₁₋₇ alkyl;hydroxy; amido (in particular where both amino substituents are H);amino (in particular where both amino substituents are H); and carboxy.

In another preferred group of compounds, the 5, 6 and 8 positions areunsubstituted (i.e. R⁵, R⁶ and R⁸=H), and the 7 position is substituted(i.e. R⁷ is not H). More preferably, the substituent (R⁷) is selectedfrom hydroxy, C₁₋₇ alkoxy (including C₁₋₇ alkyl-C₁₋₇ alkoxy and C₃₋₂₀aryl-C₁₋₇ alkoxy), and acyloxy, with C₃₋₂₀ aryl-C₁₋₇ alkoxy being themost preferred. In this group, the C₁₋₇ alkoxy is preferably eitherethoxy, especially ethoxy substituted by optionally substituted aryl (inparticular phenyl or pyridinyl), optionally substituted aryloxy (inparticular phenoxy, napthyloxy), alkoxy, sulfonoxy (in particular wherethe sulfonoxy substituent is alkyl, such as methyl or ethyl, or aryl,such as phenyl), or C₁₋₇ alkoxy is —O—CH₂—, where the alkoxy substituentis preferably optionally substituted aryl (in particular phenyl orpyridinyl) and the C₃₋₂₀ aryl group is preferably optionally substitutedphenyl, where the phenyl group being substituted is more preferred.

Preferred compounds of this type include7-methoxy-2-morpholin-4-yl-benzo[h]chromen-4-one (Compound 304),7-hydroxy-2-(morpholin-4-yl)-chromen-4-one (Compound 307),7-Benzyloxy-2-morpholin-4-yl-chromen-4-one (Compound 337),7-Benzoyloxy-2-morpholin-4-yl-chromen-4-one (Compound 423),2-Morpholin-4-yl-7-(naphthalene-2-ylmethoxy)-chromen-4-one (Compound418), 7-(4-Fluoro-benzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound414), 7-(4-Bromo-benzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound416), 7-Cyclohexylmethoxy-2-morpholin-4-yl-chromen-4-one (Compound 419),N-[3-(2-Morpholin-4-yl-4-oxo-4H-chromen-7-yloxy)-propyl]-isoindole-1,3-dione(Compound 422), 7-(2-Chloro-benzyloxy)-2-morpholin-4-yl-chromen-4-one(Compound 417), 7-(4-chlorobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one(Compound 415), 7-(4-cyano-benzyloxy)-2-morpholin-4-yl-chromen-4-one(Compound 338), 7-(3-Chlorobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one(Compound 341) and7-(3-Methylbenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 342).Of these benzyloxy-2-morpholin-4-yl-chromen-4-one (Compound 337),7-(4-Bromo-benzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound 416) and7-(4-Chlorobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 415)are particularly preferred. (See FIG. 4).

In a further preferred aspect of formula Ia, where R³ and R⁴ togetherare —A—B—, which collectively represent a fused aromatic ring which isbenzene, it is preferred that there is a further ring fused to the fusedbenzene ring, which further fused ring is preferably benzene orcyclohexane. These further fused rings may be in any position on thefused ring.

Preferred compounds of this type include2-(morpholin-4-yl)-benzo[h]chromen-4-one (Compound 293),2-(morpholin-4-yl)-benzo[g]chromen-4-one (Compound 301),7,8,9,10-tetrahydro-benzo[h]-2-(morpholin-4-yl)-chromen-4-one (Compound297), 2-(thiomorpholin-4-yl)-benzo[h]chromen-4-one (Compound 296),2-pyrrolidin-1-yl-benzo[h]chromen-4-one (Compound 312),2-morpholin-4-yl-benzo[f]chromen-4-one (Compound 310),2-(Thiazolidin-3-yl)-benzo[h]chromen-4-one (Compound 330) and2-(2-Methyl-morpholin-4-yl)-benzo[h]chromen-4-one (Compound 317), with2-(2-Methyl-morpholin-4-yl)-benzo[h]chromen-4-one (Compound 317) beingthe most preferred. (See FIG. 5).

It is generally preferred in compounds of formula Ia where R³ and R⁴together form —A—B— which represents a fused ring, that the amino groupat the 2 position (i.e. NR¹R²) is selected from dimethylmorpholino (inparticular 3,5-dimethylmorpholino), methylmorpholino (in particular3-methylmorpholino), 3,4-dihydro-2H-benzo[1,4]oxazin-4-yl,di(2-hydroxyethyl)amino, 2-(2-Hydroxy-ethoxy)-ethylamino or2-(2-Bromo-phenoxy)-ethylamino.

Preferred Aspects of Compounds of Formula Ib

For compounds of formula Ib, R⁴ is preferably H. R³ is preferably aC₅₋₂₀ aryl group, more preferably a C₅₋₂₀ carboaryl group, and inparticular an optionally substituted phenyl group. It is generallypreferred that the substituents are in the para- (or 4-)position.Preferred substituents include halo, C₁₋₇ alkyl and alkoxy, and morepreferably halo (particularly chloro) and alkoxy (particularly methoxy).

Preferred compounds of this type are6-(4-methoxyphenyl)-4-morpholin-4-yl-pyran-2-one (Compound 3) and6-(4-chlorophenyl)-4-morpholin-4-yl-pyran-2-one (Compound 4). (See FIG.1).

Preferred Aspects of Compounds of Formula Ic

In a first preferred aspect of compounds of formula Ic, R³ and R″⁴together are —A—B— which represents a fused aromatic ring which ispyridine, and the compounds are substituted at the 2-position,preferably with amino substituents. It is preferred that the aminogroups are ethylmorpholino (in particular 3-ethylmorpholino),dimethylmorpholino (in particular 3-dimethylmorpholino),2,5-dihydro-1H-pyrrol-1-yl, or pyrrolidin-1-yl.

In a second preferred aspect of compounds of formula Ic, where R³ andR′⁴ together are —A—B—, which collectively represent a fused aromaticring which is pyridine, it is preferred that a further benzene ring isfused to the pyridine (at the 7 and 8 positions) to result inpyrimidino[2,1-a]isoquinoline-4-ones. The further benzene ring ispreferably unsubstituted.

In this preferred aspect it is preferred that R¹ and R2 of formula Icform morpholine, ethylmorpholine (in particular 3-ethylmorpholine),dihydropyrrole (in particular 2,5-dihydro-1H-pyrrol-1-yl ortetrahydropyrrole).

Preferred compounds of this type are2-morpholin-1-yl-pyrimido-[2,1-a]isoquinolin-4-one (Compound 5),2-((S)-3-Hydroxy-pyrrolin-1-yl)-pyrimido[2,1-a]isoquinolin-4-one(Compound 12),2-((2S,6R)-2,6-Dimethyl-morpholin-4-yl)-pyrimido[2,1-a]isoquinolin-4-one(Compound 13) and 2-Thiomorpholin-4-yl-pyrimido[2,1-a]isoquinolin-4-one(Compound 6), with 2-morpholin-1-yl-pyrimido-[2,1-a]isoquinolin-4-one(Compound 5) being the most preferred. (See FIG. 2).

In a second preferred aspect of compounds of formula Ic, where R³ andR′⁴ together are —A—B—, which collectively represent a fused aromaticring which is pyridine, it is preferred that the 5, 6 and 8 positionsare unsubstituted (i.e. R⁵, R⁶ and R⁸=H), and that the 7 position issubstituted (i.e. R⁷ is not H). More preferably, the substituent (R⁷) isselected from hydroxy, C₁₋₇ alkoxy (including C₁₋₇ alkyl-C₁₋₇ alkoxy andC₃₋₂₀ aryl-C₁₋₇ alkoxy) and acyloxy, with C₃₋₂₀ aryl-C₁₋₇ alkoxy beingthe most preferred. In this group, the C₁₋₇ alkoxy is preferably —O—CH₂—and the C₃₋₂₀ aryl group is preferably optionally substituted phenyl.

Acronyms

For convenience, many chemical moieties are represented using well knownabbreviations, including but not limited to, methyl (Me), ethyl (Et),n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), tert-butyl (tBu),n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl(Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (Bz), andacetyl (Ac).

For convenience, many chemical compounds are represented using wellknown abbreviations, including but not limited to, methanol (MeOH),ethanol (EtOH), iso-propanol (i-PrOH), methyl ethyl ketone (MEK), etheror diethyl ether (Et₂O), acetic acid (AcOH), dichloromethane (methylenechloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF),tetrahydrofuran (THF), and dimethylsulfoxide (DMSO).

Synthesis Routes

Compounds as described in the first aspect of the invention can besynthesised by a number of methods, examples of some of which are givenbelow.

Broadly, the synthetic strategy involves performing a cyclisation toform the central core followed by a coupling reaction such as a Suzukireaction to add substituents to the core structure.

The key step in most of these synthesis routes is the formation of thecentral aromatic ring; this can be accomplished in numerous ways, asshown below, and include condensative cyclisation.

In many cases appropriate substitution can be present in the startingmaterials, although example of the further derivation of end products isalso given.

Synthesis Route 1 Synthesis of 4-Morpholin-4-yl-6-(aryl)-pyran-2-ones

(a) 3-aryl-3-hydroxy-dithioacrylic acids

A solution of CS₂ (1.81 ml, 30 mmol) and acetophenone derivative (30mmol) in dry THF (20 ml) was added dropewise over 30 min to awell-stirred solution of potassium tert-butoxide (6.73 g, 60 mmol) indry THF (50 ml) under N₂. A bright red coloration and the formation of aprecipitate were observed. The mixture was left under vigorous stirringovernight and then was poured onto water (200 ml) and extracted withether (3×100 ml). The aqueous layer was acidified with 2N H₂SO₄ to pH1-2 (Watmann pH paper) and then extracted with ether (3×100 ml). Theorganics were dried over Na₂SO₄ and the solvent was evaporated in vacuoto give the desired compound.

(b) Ethyl 3-aryl-3-hydroxy-dithioacrylates

Tetrabutylammonium hydrogen sulphate (6.76 g, 20 mmol) and sodiumhydroxide (21.6 g, 40 mmol) were dissolved in water (50 ml). A solutionof 3-aryl-3-hydroxy-dithioacrylic acid (20 mmol) in dichloromethane (50ml) was added to the solution in one portion and the reaction mixturewas stirred vigorously for 30 min. The aqueous layer was removed andiodoethane (5 ml) was added to the dichloromethane solution that wasthen stirred for 1 h. The solvent was removed in vacuo and the residuetaken into water (200 ml). The organic were extracted with ether (3×100ml), dried over Na₂SO₄ and evaporated in vacuo. The residue was thenpurified by column chromatography (ethyl acetate:petroleum ether 40-60°,1:4) to give the desired compound.

(c) 1-aryl-3-morpholin-4-yl-3-thioxo-propan-1-ones

Morpholine (1.31 ml, 15 mmol) was added to a solution of ethyl3-aryl-3-hydroxy-dithioacrylate (15 mmol) in ethanol (20 ml). Thereaction mixture was refluxed for 5 h and upon cooling at roomtemperature the desired compound crystallized. The compound was thenisolated by filtration.

(d) 1-aryl-3-ethylsulfanyl-3-morpholin-4-yl-propen-1-ols

1-aryl-3-morpholin-4-yl-3-thioxo-propan-1-one (12 mmol) was dissolved indry acetone (20 ml) and finely powdered K₂CO₃ (1.83 g, 13.2 mmol) andiodoethane (1.07 ml, 13.2 mmol) were added to the solution. The reactionmixture was then reflux overnight and the solvent was then removed invacuo. The residue was taken into water (50 ml) and the organics wereextracted with dichloromethane (3×30 ml), dried over sodium sulfate andevaporated in vacuo. The residue was purified by column chromatographyto give the desired compound.

(e) 4-morpholin-4-yl-6-(aryl)-pyran-2-ones

A suspension of activated zinc (heated at 120° C. for 1 hr) (2.6 g, 0.04g atom), ethyl bromoacetate (3.18 g, 20 mmol) and a few crystals ofiodine in dry THF (30 ml) was heated at 50° C. for 45 min with stirring.A solution of the respective1-aryl-3-ethylsulfanyl-3-morpholin-4-yl-propenone (10 mmol) in dry THF(50 ml) was added dropwise with stirring and the mixture was refluxedfor 3-4 h. The mixture was then poured over-ice cold dilute 3% H₂SO₄(100 ml), the aqueous layer was extracted with ethyl acetate (3×50 ml),the combined extract was dried over Na₂SO₄ and the solvent wasevaporated. The residue was purified by column chromatography (ethylacetate:pet ether 40-60, 1:4) to give the pure pyran-2-one.

Variations

If the amino group in the final product is desired to be other thanmorpholino, than the relevant amine can be used in step (c) in place ofmorpholine. The 6-aryl group in the final product can be a heteroarylgroup, if the appropriate acetophenone derivative is used as a startingmaterial.

Synthesis Route 2 Synthesis of 2-Amino pyrimidine isoquinolin-4-ones

References: Snyder and Robison, J. Amer. Chem. Soc., 74; 4910-4914(1952); Di Braccio, M., et al., Eur. J. Med. Chem.; 30(1), 27-38 (1995).

(a) Pyrimido[1,2-a]isoquinoline-2,4-dione

Aminoisoquinoline (5.16 g, 35.79 mmol) was dissolved in diethyl malonate(5.43 ml, 35.79 mmol). Ethanol (20 ml) was added, and the solution washeated to 170° C. for 4 h. The ethanol was removed by distillation andupon cooling, the dark residue in the reaction flask was triturated inethyl acetate (10 ml). This resulted in formation of a pale solid whichwas collected by filtration and washed with ethyl acetate to furnish thetitle compound as a pale brown solid. (4.43 g, 24.89 mmol, 70% yield).mp=294-296° C. Analytically pure by LC-MS: m/z (ES⁺): 213 (M⁺)

(b) 2-Chloro-pyrimido[1,2-a]isoquinolin-4-one

Pyrimido[1,2-a]isoquinoline-2,4-dione (4.43 g, 24.89 mmol) was dissolvedin phosphorous oxychloride (20 ml) and this solution was heated toreflux for 5 h. Upon cooling, the reaction mixture was poured carefullyinto ice water (˜250 ml) and adjusted to pH 7 by addition of sodiumcarbonate. This resulted in formation of a brown precipitate which wascollected by filtration and washed with water to yield a brown solid.The crude product was chromatographed, eluting with DCM to provide thetitle compound as pale yellow crystals. (5.21 g, 22.70 mmol, 91% yield).mp 197-199° C. Analytically pure by LC-MS: m/z (ES⁺): 231.5 (M⁺)

(c) 2-Aminopyrimidine isoquinolin-4-ones

2-Chloro-pyrimido[2,1-a]isoquinolin-4-one was dissolved in boilingethanol (20 ml), and to this solution was added the appropriate amine (4mol equiv). The solution was heated to reflux, with vigorous stirring,for 16 h. The reaction mixture was then allowed to cool to roomtemperature, upon which a solid slowly crystallised. The crystallinesolid was collected by filtration and washed with cold ethanol (30 ml).This solid was dried under vacuum to provide the desired compound.

Variations

If different substituents are desired on the central core of two fusedrings, these can be introduced by varying the substituents on the2-amino pyridine ring of the starting material, using protecting groupswhere appropriate.

Synthesis Route 3 Synthesis of 2-Chloro-6-morpholin-4-yl-pyran-4-ones

(a) 4-Chloro-4-(2,2,2-trichloro-ethyl)-oxetan-2-one

A solution of (bis-4-t-butylcyclohexyl) peroxydicarbonate (11.8 g) anddiketene (83.5 ml) in CCl₄ (300 ml) was added drop wise over 120 min toa refluxing solution of CCl₄, and was stirred for a further 1 h. Theresulting pale yellow solution was cooled and azeotroped withdichloromethane. The resulting residue was stirred with hexane (3×150ml) for 10 min and the liquor was decanted off through a celite pad. Thefiltered liquors were combined and concentrated in vacuo to give thedesired compound as a pale yellow oil (125.0 g, 52.9%).

(b) 5,5-Dichloro-1-morpholin-4-yl-pent-4-ene-1,3-dione

Two separate solutions of4-Chloro-4-(2,2,2-trichloro-ethyl)-oxetan-2-one (62.5 g, 0.26 mmol) andmorpholine (24.0 g, 0.28 mol) in dichloromethane (120 ml) were addedsimultaneously to a mixture of NaHCO₃ (44.0 g, 0.52 mol) in drydichloromethane (300 ml). The reaction was maintained at 15° C. over 140min with stirring. The reaction was filtered, washed withdichloromethane (3×100 ml) and the combined organic layers wereconcentrated in vacuo to a slurry which was then passed through a shortsilica pad, and further washed with dichloromethane (4×100 ml). Thecombined organic layers were concentrated in vacuo, suspended in hexane(400 ml) and stirred for 1 h, filtered and dried to give a cream solid.The solid was suspended in tert-butyl methyl ether (100 ml), stirred for15 min, filtered, washed with butyl methyl ether and dried to give thedesired compound as a white powder (47.8 g, 72%). m/z (LC-MS, ESP): 252(M⁺+1).

(c) 2-Chloro-6-morpholin-4-yl-pyran-4-one

To a suspension of 5,5-Dichloro-1-morpholin-4-yl-pent-4-ene-1,3-dione(11.3 g, 44.9 mmol) in dioxane was added perchloric acid (11.4 ml, 0.14mol) and the reaction was heated at 90° C. under N₂ for 1 h. Thereaction was cooled, neutralised with 2M NaOH (75 ml) and filtered. Theaqueous layer was extracted with dichloromethane (4×30 ml) and theorganic layers were combined and dried over MgSO₄. The organic layer wasfurther treated with charcoal and filtered through celite. The darkyellow filtrate was evaporated in vacuo, and the resulting solid wastriturated with hexane (50 ml) and dried to give the desired compound(7.3 g, 75%) as a light yellow powder. m/z (LC-MS, ESP): 216 (M⁺+1).¹HNMR (300 MHz, DMSO-d₆): 3.3 (t, 4H), 3.65 (t, 4H), 5.4 (d, 1H), 6.25(d, 1H).

Variations:

If the amino group in the final product is desired to be other thanmorpholino, then the relevant amine, for example dimethylmorpholine canbe used in step (b) in place of morpholine.

Synthesis Route 4 Synthesis of 6-Aryl-2-morpholin-4-yl-pyran-4-one and6-heterocycle-2-morpholin-4-yl-pyran-4-one

(a) 6-Aryl-2-morpholin-4-yl-pyran-4-ones

A solution of chloropyranone (22 mg, 0.1 mmol) in dioxane (0.3 ml,degassed by sonication and saturation with N₂) was added to aryl boronicacid (0.13 mmol) and Cs₂CO₃ (65 mg, 0.2 mmol) under N₂ atmosphere.Pd(PPh₃)₄ (5 mg, 0.005 mmol) in dioxane (0.2 ml, degassed by sonicationand saturation with N₂) was then added to the solution under N₂atmosphere. The reaction was heated at 90° C. with vigorous stirringovernight. The sample was diluted with methanol/dichloromethane (1:2; 1ml), passed through a plug of silica (isolute Si 500 mg) and purified bypreparative HPLC.

Variations

Where the 6-substituent is desired to be a heterocycle rather than aryl,the appropriate heterocycle boronic acid can be substituted for arylboronic acid above.

Synthesis Route 4a Synthesis of N-Alkyl3-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzamide Derivatives

(a) 3-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid methyl ester

2-Chloro-6-morpholin-4-yl-pyran-4-one (7.98 g, 37 mmol),(3-methoxycarbonylphenyl) boronic acid (8.01 g, 44.5 mmol), and groundpotassium carbonate (11.23 g, 81.40 mmol) were suspended in dioxane (50ml) and degassed (sonication for 5 min then saturated with N₂).Pd(PPh₃)₄ (2.13 g, 1.85 mmol) was then added and the reaction mixturewas then heated at 90° C. for 24 hrs under a vigorous stirring and a N₂atmosphere. The solvent were removed in vacuo and the residue was thensuspended in water 50 ml) and extracted with ethyl acetate (100 ml). Theorganics were combined, washed with saturated brine and dried oversodium sulphate. The solvent was removed in vaccuo and the residue waspurified by column chromatography (silica; dichloromethane:methanol;9:1) to give the title compound as a white solid (5.42 g, 46%). m/z(LC-MS, ESP): 316 (M⁺+1).

(b) 3-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid sodium salt

3-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid methyl ester (5.42g, 17.20 mmol) was dissolved in methanol (25 ml) and sodium hydroxide(0.75 g, 18.90 mmol) was added. The stirred solution was then refluxedunder nitrogen for three hours. The methanol was removed in vacuo andthe residue was triturated in ether to give the title compound as abrown solid (4.30 g, 83.33%). m/z (LC-MS, ESP): 301 (M⁺+1).

(c) N-Alkyl 3-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzamideDerivatives

To a stirred solution of3-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid sodium salt (52mg, 0.16 mmol) in anhydrous dimethylacetamide (1 ml),N,N-dimethylaminopyridine (2 mg, catalytic) and ethylchloroformate (19μl, 0.192 mmol) were added, the solution was stirred for 45 minutes. Thedesired amine (0.32 mmol) was then added to the reaction mixture wasleft under stirring overnight. The compound was then purified bypreparative HPLC to give the desired compound.

Variations

Where an aryl other than phenyl, or a heterocycle, is desired at the3-position, the appropriate (methoxycarbonylaryl/heterocycle) boronicacid is substituted for (3-methoxycarbonylphenyl) boronic acid in step(a).

Synthesis Route 4b Synthesis of N-Alkyl4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzamide Derivatives

(a) 4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid methyl ester

2-Chloro-6-morpholin-4-yl-pyran-4-one (4.01 g, 18.60 mmol),(4-methoxycarbonylphenyl)boronic acid (4.01 g, 22.32 mmol), and groundpotassium carbonate (5.64 g, 40.92 mmol) were suspended in dioxane (20ml) and degassed (sonication for 5 min then saturated with N₂).Pd(PPh₃)₄ (0.5 g, 0.4 mmol) was then added and the reaction mixture wasthen heated at 90° C. for 24 hrs under a vigorous stirring and a N₂atmosphere. The solvent were removed in vacuo and the residue was thensuspended in water 50 ml) and extracted with ethyl acetate (100 ml). Theorganics were combined, washed with saturated brine and dried oversodium sulphate. The solvent was removed in vacuo and the residue waspurified by column chromatography (silica; dichloromethane:methanol;9:1) to give the title compound as a white solid 3.71 g, 63%). m/z(LC-MS, ESP): 316 (M⁺+1).

(b) 4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid sodium salt

4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid methyl ester (3.00g, 9.52 mmol) was dissolved in methanol (20 ml) and sodium hydroxide(0.381 g, 9.52 mmol) was added. The stirred solution was then refluxedunder nitrogen for three hours. The methanol was removed in vacuo andthe residue was triturated in ether to give the title compound as abrown solid (3 g, 97%). m/z (LC-MS, ESP): 301 (M⁺+1).

(c) N-Alkyl 4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzamideDerivatives

To a stirred solution of4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-benzoic acid sodium salt (52mg, 0.16 mmol) in anhydrous dimethylacetamide (1 ml),N,N-dimethylaminopyridine (2 mg, catalytic) and ethylchloroformate (19μl, 0.192 mmol) were added, the solution was stirred for 45 minutes. Thedesired amine (0.32 mmol) was then added to the reaction mixture wasleft under stirring overnight. The compound was then purified bypreparative HPLC to give the desired compound.

Variations

Where an aryl other than phenyl, or a heterocycle, is desired at the4-position, the appropriate (methoxycarbonylaryl/heterocycle) boronicacid is substituted for (4-methoxycarbonylphenyl) boronic acid in step(a).

Synthesis route 4c(i) Synthesis of(3-aminomethyl-phenyl)-6-morpholin-4-yl-pyran-4-one Derivatives

(a) [3-(6-Morpholine-4-yl-4-oxo-4H-pyran-2yl)-phenyl]benzaldehyde

Chloropyranone (10.75 g, 50 mmol) and 3-formylphenylboronic acid (9.0 g,60 mmol) were stirred in a solution of degassed dioxane (110 ml) for 20min. This was followed by the addition of Na2CO3 (13.8 g, 100 mmol) andtetrakis(triphenylphosphine) palladium (2.88 g, 2.5 mmol). The reactionmixture was further degassed for 10 min and heated to 80° C. under N₂for 18 h. The reaction was then cooled to room temperature, concentratedin vacuo and purified by flash column chromatography (ethylacetate/methanol) to yield3-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)benzaldehyde as a orange solid(6.5 g, 45%). m/z (LC-MS, ESP): 286 (M⁺+1).

(b) (3-aminomethyl-phenyl)-6-morpholin-4-yl-pyran-4-ones Derivatives

3-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)benzaldehyde (0.2 mmol) and theappropriate amine (0.24 mmol) were dissolved in dichloroethane (2 ml).Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0mmol) were then added and stirred at room temperature for 16 h. Thereaction mixtures were then purified by preparatory HPLC.

Synthesis route 4c(ii) Synthesis of(4-aminomethyl-phenyl)-6-morpholin-4-yl-pyran-4-one Derivatives

(a) [4-(6-Morpholine-4-yl-4-oxo-4H-pyran-2yl)-phenyl]benzaldehyde

Chloropyranone (10.75 g, 50 mmol) and 4-formylphenylboronic acid (9.0 g,60 mmol) were stirred in a solution of degassed dioxane (110 ml) for 20min. This was followed by the addition of Na₂CO₃ (13.8 g, 100 mmol) andtetrakis(triphenylphosphine) palladium (2.88 g, 2.5 mmol). The reactionmixture was further degassed for 10 min and heated to 80° C. under N₂for 18 h. The reaction was then cooled to room temperature, concentratedin vacuo and purified by flash column chromatography (ethylacetate/methanol) to yield4-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)benzaldehyde as a yellow powder(6 g, 42%). m/z (LC-MS, ESP): 286 (M⁺+1).

(b) (4-aminomethyl-phenyl)-6-morpholin-4-yl-pyran-4-ones Derivatives

4-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)benzaldehyde (0.2 mmol) and theappropriate amine (0.24 mmol) were dissolved in dichloroethane (2 ml).Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0mmol) were then added and stirred at room temperature for 16 h. Thereaction mixtures were then purified by preparatory HPLC.

Synthesis route 4d (i) Synthesis of(3-amino-phenyl)-6-morpholin-4-yl-pyran-4-ones

(a) Synthesis of[3-(6-Morpholine-4-yl-4-oxo-4H-pyran-2-yl)-phenyl]carbamic acidtert-butyl ester

Chloropyranone (1.8 g, 8.35 mmol) and 3-(BOC-aminophenyl)boronic acid(2.4 g, 10 mmol) were stirred in a solution of degassed dioxane (45 ml)for 20 min. This was followed by the addition of Na₂CO₃ (2.78 g, 20.16mmol) and tetrakis(triphenylphosphine) palladium (483 mg, 0.08 mmol).The reaction mixture was further degassed for 10 min and heated to 80°C. under N₂ for 18 h. The reaction was then cooled to room temperature,concentrated in vacuo and purified by flash column chromatography (ethylacetate/methanol) to yield to the title compound (1.51 g, 48%). m/z(LC-MS, ESP): 373 (M⁺+1).

(b) Synthesis of (3-amino-phenyl)-6-morpholin-4-yl-pyran-4-one

[3-(6-Morpholine-4-yl-4-oxo-4H-pyran-2yl)-phenyl]carbamic acidtert-butyl ester (3.4 g, 9.2 mmol) was dissolved in 25% trifluoroaceticacid in dichloromethane mixture (30 ml) and stirred for 1 hour at roomtemperature. The reaction was concentrated in vacuo, precipitated withsaturated NaHCO₃, filtered, washed with diethyl ether and dried to yield(3-amino-phenyl)-6-morpholin-4-yl-pyran-4-one as a white solid (2.1 g,85%). m/z (LC-MS, ESP): 273 (M⁺+1).

Synthesis route 4d (ii) Synthesis of(4-aminophenyl)-6-morpholin-4-yl-pyran-4-ones

(a) Synthesis of[4-(6-Morpholine-4-yl-4-oxo-4H-pyran-2yl)-phenyl]carbamic acidtert-butyl ester

Chloropyranone (1 g, 4.64 mmol) and 4-(BOC-aminophenyl)boronic acid(1.14 g, 5.57 mmol) were stirred in a solution of degassed dioxane (10ml) for 20 min. This was followed by the addition of Na₂CO₃ (1.41 g,10.21 mmol) and tetrakis(triphenylphosphine) palladium (268 mg, 0.05mmol). The reaction mixture was further degassed for 10 min and heatedto 80° C. under N₂ for 18 h. The reaction was then cooled to roomtemperature, concentrated in vacuo and purified by flash columnchromatography (ethyl acetate/methanol) to yield to the title compound(0.9 g, 52%). m/z (LC-MS, ESP): 373 (M⁺+1).

(b) Synthesis of (4-amino-phenyl)-6-morpholin-4-yl-pyran-4-one

[4-(6-Morpholine-4-yl-4-oxo-4H-pyran-2yl)-phenyl]carbamic acidtert-butyl ester (402 mg, 1.08 mmol) was dissolved in 25%trifluoroacetic acid in dichloromethane mixture (5 ml) and stirred for 1hour at room temperature. The reaction was concentrated in vacuo,precipitated with saturated NaHCO₃, filtered, washed with diethyl etherand dried to yield (4-amino-phenyl)-6-morpholin-4-yl-pyran-4-one as ayellow solid (230 mg, 79%). m/z (LC-MS, ESP): 273 (M⁺+1).

Synthesis route 4d(iii)(4-acylamido-phenyl)-6-Morpholin-4-yl-pyran-4-ones Derivatives

(a) Appropriate acid chloride (0.24 mmol) was added to a solution of(4-Amino-phenyl)-6-morpholin-4-yl-pyran-4-one (0.2 mmol) indichloromethane (2 ml). Hünig's base (0.4 mmol) was then added and thereaction was stirred at room temperature for 16 h. The reaction mixtureswere then purified by preparatory HPLC.

Variations

Isocyanate or isothiocyanate can be used in place of acid chloride togenerate ureido or thioureido structures.

Synthesis route 4d(iv)(3-acylamido-phenyl)-6-Morpholin-4-yl-pyran-4-ones Derivatives

(a) Appropriate acid chloride (0.24 mmol) was added to a solution of(3-Amino-phenyl)-6-morpholin-4-yl-pyran-4-one (0.2 mmol) indichloromethane (2 ml). Hünig's base (0.4 mmol) was then added and thereaction was stirred at room temperature for 16 h. The reaction mixtureswere then purified by preparatory HPLC.

Variations

Isocyanate or isothiocyanate can be used in place of acid chloride togenerate ureido or thioureido structures.

Synthesis route 4d(v) Synthesis of(3-amino-phenyl)-6-morpholin-4-yl-pyran-4-one Derivatives

(a) (3-Amino-phenyl)-6-morpholin-4-yl-pyran-4-one (0.2 mmol) and theappropriate aldehydes (0.24 mmol) were dissolved in dichloroethane (2ml). Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid(6.0 mmol) was then added and stirred at room temperature for 16 h. Thereaction mixtures were then purified by preparatory HPLC.

Synthesis Route 5 Synthesis of 2-(4-Morpholinyl)-6-aryl-4H-pyran-4-ones

(a) 3-(Aryl)-3-oxo-2-triphenylphosphoranylpropionates

A mixture of methyl triphenylphosphoranylideneacetate (20 mmol) andappropriate aroyl chloride (10 mmol) in anhydrous toluene (100 ml) undernitrogen was refluxed for 3 h, cooled to room temperature and the whiteprecipitate formed was filtered. The filter cake was thoroughly washedwith ethyl acetate (4×40 ml) and the combined filtrate evaporated invacuo. The oil was purified by column chromatography to give the desiredcompound.

(b) Methyl 3-(aryl)propiolates

Methyl 3-(3-aryl)-3-oxo-2-triphenylphosphoranylpropanoate (9 mmol) wasslowly warmed to 250° C. in a kugelrohr distillation apparatus (1 Torr).Distillate was collected for 20 minutes at 250° C. and was purified bycolumn chromatography to give the desired product.

(c) 4-[(2-Oxo-4-aryl-3-butynyl)carbonyl]morpholine lithium salts

n-Butyllithium (2.5 M in hexanes, 5.3 ml, 13.2 mmol) was added dropwiseat 0° C. to a stirred solution of diisopropylamine (1.87 ml, 13.2 mmol)in THF (20 ml) under a nitrogen atmosphere. After 30 minutes, acetylmorpholine (1.53 ml, 13.2 mmol) was added dropewise to the reactionmixture and left for one h under stirring at 0° C. The reaction was thencooled to −78° C. and methyl 3-(3-aryl)propiolate (6 mmol) in THF (5 ml)was added dropwise to the reaction mixture and left to react at −78° C.for 30 minutes and then to 0° C. for 1 h. The reaction mixture wasquenched with water (15 ml) and the white suspension extracted twicewith dichloromethane (30 ml). The organics were combined and evaporatedunder reduce pressure to give a solid which was triturated with acetone(10 ml). The solid was filtered and washed successively with water (5ml), acetone (5 ml) and ether (5 ml). The solid obtained was then driedin vacuo overnight at 40° C. to give the desired compound.

(d) 2-(4-Morpholinyl)-6-aryl-4H-pyran-4-one

A solution of 4-[(2-oxo-4-aryl-3-butynyl)carbonyl]morpholine lithiumsalt (2 mmol) in methanesulphonic acid (6 ml) was stirred under nitrogenfor 3 h at room temperature. The mixture was poured into saturatedsodium carbonate solution (100 ml) and extracted with dichloromethane(3×50 ml). The combined organics were dried over sodium sulphate andevaporated in vacuo. The residue was purified by column chromatography

Variations

If the amino group in the final product is desired to be other thanmorpholino, than the relevant acetyl amine can be used in step (c) inplace of acetyl morpholine.

Synthesis Route 6 Synthesis of 2-amino-chromen-4-ones (1^(st) Method)

(a) Salicylate Esters

A solution of the appropriate acid in methanol (150 ml) was treated withconcentrated sulphuric acid (3 ml). The solution was heated to refluxfor 40 h and then cooled to room temperature. The reaction mixture wasevaporated in vacuo and then re-suspended in ethyl acetate (200 ml). Thesolution was washed with 50% saturated sodium bicarbonate solution(4×150 ml). The aqueous extracts were combined and washed with ethylacetate (150 ml). The organic extracts were combined, washed with brine(50 ml), dried over sodium sulphate and evaporated in vacuo to give theproduct, which was then crystallised from methanol to provide thedesired compound.

(b) β-ketoamides

A solution of diisopropylamine (5.1 ml, 3.0 mmol) in THF (30 ml) wascooled to −70° C. and slowly treated with 2.5 M solution of n-butyllithium in hexanes (14.0 ml, 35 mmol) and then warmed to 0° C. andstirred for 15 minutes. The solution was cooled to −10° C. and slowlytreated with a solution of N-acetyl morpholine, N-acetyl piperidine, orN-acetyl thiomorpholine in THF (25 ml), maintaining the temperaturebelow −10° C. The reaction mixture was stirred at this temperature for90 minutes and then treated with a solution of the relevant salicylateester in THF (25 ml), followed by additional THF (5 ml). The reactionmixture was slowly warmed to room temperature and stirred for 16 h. Thesolution was quenched with water (5 ml) and 2 M hydrochloric acid (50ml) and extracted into DCM (3×80 ml). The organic extracts werecombined, washed with brine (50 ml), dried over sodium sulphate andevaporated in vacuo to give an oily residue. The crude product wasstirred vigorously in hot ether, causing precipitation of a white solid.This was collected, after cooling in ice, by filtration and washed withcold ether, to provide the desired compound.

(c) 2-amino-chromen-4-ones

A solution of the appropriate β-ketoamides in DCM (35 ml) was treatedwith triflic anhydride (3.8 ml, 23 mmol) and stirred at room temperatureunder nitrogen for 16 h. The mixture was evaporated in vacuo and thenre-dissolved in methanol (80 ml). The solution was stirred for 4 h,treated with water (80 ml) and stirred for a further hour. The mixturewas evaporated in vacuo to remove methanol. The aqueous mixture wasadjusted to pH 8 by treatment with saturated sodium bicarbonate and thenextracted into DCM (3×150 ml). The extracts were dried over sodiumsulphate and evaporated in vacuo to give a solid. The crude product waspartially dissolved in DCM and loaded onto a silica column, eluting withDCM followed by (1%; 2%; 5%) methanol in DCM. All fractions containingthe desired product were combined and evaporated in vacuo to give anorange solid. The crude product was dissolved in hot methanol, treatedwith charcoal, filtered through celite and recrystallised from methanolto provide the desired compound.

Variations

If the amino group in the final product is desired to be other thanmorpholino, than the relevant acetyl amine can be used in step (b) inplace of acetyl morpholine.

Synthesis Route 7a Synthesis of 2-amino-chromen-4-ones (2^(nd) Method)

Di Braccio, M., et al., Farmaco, 50(10), 703-711 (1995); Vlahos, C. J.,et al., J. Biol. Chem., 269(7), 5241-5248 (1994).

(a) 4-hydroxy-chromen-2-thiones

A suspension of potassium tert-butoxide (7.20 g, 64 mmol) in toluene (50ml) was cooled to ˜10° C. and treated with a solution of the appropriateacetoaryl and carbon disulphide (1.20 ml, 20.0 mmol) in toluene (50 ml).The resultant mixture was stirred at room temperature for 16 h and thentreated with water (500 ml). The mixture washed with ether (2×100 ml)and charged into a 3-neck round bottom flask. The aqueous solution wastreated with 10% sulphuric acid, venting the flask through a bleachtrap. The resultant suspension was stirred for 24 h to allow for removalof hydrogen sulphide. The solid was collected by filtration, washingwith water (3×50 ml) and cold petrol (3×50 ml). Recrystallisation fromethyl acetate/petrol provided the desired compound.

(b) 2-(Ethylthio)-chromen-4-ones

A solution of 4-hydroxy-chromen-2-thione in acetone (10 ml) is treatedwith potassium carbonate and ethyl iodide and heated to reflux. Thereaction mixture was evaporated in vacuo, re-dissolved in DCM (20 ml)and washed with water (20 ml). The aqueous layer washed with additionalDCM (3×20 ml) and the organic extracts were combined, dried over sodiumsulphate and evaporated in vacuo. The residue was recrystallised fromethyl acetate/petrol to provide the desired compound.

(c) 2-amino-benzo-chromen-4-ones

A solution of the appropriate 2-(ethylthio)-benzo-chromen-4-one in DCM(10 ml) at 0° C. is treated with a solution of mCPBA in DCM (10 ml) andstirred at room temperature. The reaction mixture is cooled to −20° C.to form a precipitate which is removed and washed. This is suspended inacetonitrile, and treated with the appropriate secondary amine andstirred at room temperature. The reaction mixture is evaporated in vacuoand re-dissolved in ethyl acetate (100 ml). This solution is then washedwith 50% saturated sodium bicarbonate solution (2×100 ml), dried oversodium sulphate and evaporated in vacuo. The solid residue is trituratedin ether, filtered and the solid collected recrystallised from methanolto provide the desired compound.

(d) 2-amino benzenechromen-4-ones

A mixture of 2-ethylsulphanyl-benzochromen-4-one, the appropriate amine(10 mol equiv) and ethylene glycol (10 ml) was heated to 160° C., withstirring, for 3 h. Upon cooling to room temperature the reaction mixturewas poured onto ice water (100 ml) and extracted into DCM. The organicextracts were collected, dried over sodium sulphate, and the solvent wasremoved by evaporation in vacuo to yield the product as a pale solid.The product was purified by recrystallisation from a suitable solvent.

(e) (Benzo-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneResin

Merrifield resin (1% cross-linked, 1.2 mmol/g) (0.70 g, 0.84 mmol) wasswelled in anhydrous DMF (4 ml). The mixture was shaken gently for 15minutes and then treated with a solution of the appropriate4-Hydroxy-benzo-chromen-2-thione (0.50 g, 2.2 mmol) in DMF (3 ml). Aftershaking for a further 15 minutes, the mixture is treated with1,8-diazabicyclo[5.4.0]undec-7-ene (0.4 ml, 2.7 mmol). The reactionmixture is then heated to 70° C. and gently shaken for 24 h. The resinis collected by filtration and washed with DMF, followed by methanol andfinally washed with DCM.

(f) Benzo-chromen-4-ones Library

The appropriate(Benzo-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzene resin(0.030 g, 0.036 mmol) is swelled in anhydrous DMF and gently shaken for15 minutes. The reaction mixture was treated with a prepared solution ofamine (0.036 mmol) in DCM (0.2 ml). The mixture is shaken at roomtemperature for 24 h, followed by addition of Amberlite IR120+ resin (50mg) and shaking for a further 1 h. The reaction mixture is thenfiltered, washing the resin with DCM and methanol. The filtrate wasevaporated in vacuo to provide 0.0014 g (0.004 mmol) of the crudedesired compound, which is submitted for analysis by LC-MS withoutfurther purification.

Variations

If different substituents are desired on the central core of two fusedrings, these can be introduced by varying the substituents on the ringof the salicylic acid starting material, using protecting groups whereappropriate (e.g. see route 7b).

Substituted Morpholines

Substituted morpholines such as 2-Ethyl-morpholine and2,2-Dimethyl-morpholine were prepared using methodology described inBettoni et al. Tetrahedron, 1980, 36, 409-415, as discussed in relationto Compound 317 below.

Synthesis Route 7b Solid Phase Synthesis of7-alkoxy-2-(morpholin-4-yl)-chromen-4-ones

Steps (a), (e) and (f) are as for Synthesis Route 7a.

(g)(7-(Alkoxyoxy)-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneResins

(7-(Hydroxy)-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneresin (0.030 g, <0.036 mmol) was swelled in anhydrous DMF and gentlyshaken for 15 minutes. The mixture was treated with1,8-diazabicyclo[5.4.0]undec-7-ene (0.2 ml, 1.3 mmol). After shaking fora further 15 minutes, the mixture was treated with an alkylating agent(e.g. benzyl bromide). The reaction was heated to 65° C. and shaken for20 h. The resin was collected by filtration and washed in order withDMF, methanol and DCM. This procedure was repeated on the resin withfresh reagents a further 3 times.

Variations

If different substituents are desired on the central core of two fusedrings, these can be introduced by varying the substituents on the ringof the acetophenone starting material, for example using2,5-dihydroxyacetophenone in place of 2,4-dihydroxyacetophenone togenerate 6-hydroxy substituted chromen-4-ones.

Synthesis Route 7b(i) Derivatisation of 7-hydroxy Substitutedchromen-4-ones

(a)(7-aryloxy-4-oxo-4H-chromen-7-yl)-thiomethylpolystyrene-divinylbenzeneresins

S-(7-Hydroxy-4-oxo-4H-chromen-7-yl)-thiomethylpolystyrene-divinylbenzeneresin (0.020 g, <0.024 mmol) was swelled in THF (1 ml) in an AdvancedChemtech reaction fritted vessel and gently shaken for 15 min. Gentlyagitating for 10 min between the addition of each reagent, the vesselwas sequentially treated with TEA (0.05 ml), a solution oftriphenylphosphine (0.063 g) in THF (0.5 ml) and a solution of theappropriate alcohol (0.25 mmol) in THF (0.5 ml). After a further 10 minthe vessel was treated with a solution of DIAD (0.047 ml) in THF (0.5ml), chilled in a dry ice/acetone bath prior to addition. The reactionvessels were gently agitated for 20 h, drained, and the resin washedwith DCM×2, DMF×1, methanol×1 and DCM×2

(b) 7-aryloxy-2-morpholin-4-yl-chromen-4-ones

The resin bound chromone (maximum 0.036 mmol) was suspended in DCM (2ml) and after shaking for 10 min, the mixture was treated with mCPBA(0.2 g, 1.1 mmol). The mixture was shaken at room temperature for 3hours and then filtered. The resin washed in order with DCM×2,methanol×2, DCM×2 and re-suspended in DCM (2 ml). After shaking for 15minutes the mixture was treated with a solution of morpholine (0.005 ml,0.05 mmol) in DCM (2 ml). The mixture was shaken at room temperature for16 h and filtered, washing the resin with methanol (2×2 ml). Thefiltrate was evaporated in vacuo to provide the title compound. Theproduct was submitted for analysis by LC-MS without furtherpurification.

Variations

If the amino group in the final product is desired to be other thanmorpholino, than the amine can be used in step (b) in place ofmorpholine. The 7-substituent may be substituted or unsubstituted alkyl,heterocyclyl, etc rather than aryl by using the appropriate alcohol instep (a).

Synthesis Route 7c Synthesis of 2-(morpholin-4-yl)-chromen-4-onesDerivatives

(a) Aryl substituted 2-(morpholin-4-yl)-chromen-4-ones

Organoboron compound (0.058 mmol), Trifluoro-methanesulfonic acid2-morpholin-4-yl-4-oxo-4H-chromenyl ester (Compound 305 or 306) (20 mg,0.053 mmol and powdered potassium carbonate (14.6 mg, 0.106 mmol) wereadded to a reaction tube, which was then purged with nitrogen andsealed. A flask of dioxane was degassed with nitrogen purge andsonication for 5 min before addition to the reaction tube (0.5 ml). Tothis was added a solution of tetrakis(triphenylphosphine) palladium(0)(3.1 mg) in degassed dioxane (0.3 mL) and the reaction mixture washeated to 90° C. with reflux under a nitrogen atmosphere for 18 h. Thereaction was cooled and passed through a silica plug (isolute Si 500 mgcartridge) and eluted with 30% Methanol/DCM (8 mL). The solution wasanalysed by LCMS and purified by preparative HPLC.

Synthesis Route 8 Further Derivitisation of7-(hydroxy)-2-(morpholin-4-yl)-chromen-4-one to7-alkoxy-2-(morpholin-4-yl)-chromen-4-ones

A solution of 7-(hydroxy)-2-(morpholin-4-yl)-chromen-4-one (307) (0.125g, 0.50 mmol) in anhydrous DMF (5 ml) was treated with the appropriatearyl bromide, followed by a 40% methanolic solution ofbenzyltrimethylammonium hydroxide (0.54 ml, 1.2 mmol). The solution washeated to 80° C. and stirred for 16 h. After cooling, the solution wastreated with ethyl acetate (25 ml) and water (10 ml). The mixture wasstirred vigorously for 30 minutes and allowed to settle. The ethylacetate layer was removed by pipette and evaporated in vacuo. The crudeproduct was recrystallised from methanol.

Synthesis Route 9 Further Derivitisation of7-(hydroxy)-2-(morpholin-4-yl)-chromen-4-one to7-aroyloxy-2-(morpholin-4-yl)-chromen-4-ones

A solution of 7-hydroxy-2-(morpholin-4-yl)-chromen-4-one (299)(0.25 g,1.0 mmol) in DMF (10 ml) was treated with the appropriate aroylchloride, followed by pyridine (0.10 ml, 1.2 mmol) at 0° C. The solutionwas warmed to room temperature and stirred for 16 h. The resultantsuspension was diluted with ethyl acetate (100 ml) and washed with 0.5 Mhydrochloric acid (50 ml), water (50 ml) and brine (50 ml). The organicextract was dried over sodium sulphate and evaporated in vacuo. Thecrude product was recrystallised from ethyl acetate.

Use of Compounds of the Invention

The present invention provides active compounds, specifically, active4-amino-pyran-2-ones, 2-amino-pyran-4-ones, 2-amino-4-ones, and2-amino-pyridine-isoquinolin-4-ones.

The term “active”, as used herein, pertains to compounds which arecapable of inhibiting DNA-PK activity, and specifically includes bothcompounds with intrinsic activity (drugs) as well as prodrugs of suchcompounds, which prodrugs may themselves exhibit little or no intrinsicactivity.

One assay which may be used in order to assess the DNA-PK inhibitionoffered by a particular compound is described in the examples below.

The present invention further provides a method of inhibiting DNA-PKinhibition in a cell, comprising contacting said cell with an effectiveamount of an active compound, preferably in the form of apharmaceutically acceptable composition. Such a method may be practisedin vitro or in vivo.

For example, a sample of cells (e.g. from a tumour) may be grown invitro and an active compound brought into contact with said cells inconjunction with agents that have a known curative effect, and theenhancement of the curative effect of the compound on those cellsobserved.

The present invention further provides active compounds which inhibitDNA-PK activity as well as methods of methods of inhibiting DNA-PKactivity comprising contacting a cell with an effective amount of anactive compound, whether in vitro or in vivo.

The invention further provides active compounds for use in a method oftreatment of the human or animal body. Such a method may compriseadministering to such a subject a therapeutically-effective amount of anactive compound, preferably in the form of a pharmaceutical composition.

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g. in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,and cure of the condition. Treatment as a prophylactic measure (i.e.prophylaxis) is also included.

The term “therapeutically-effective amount” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio.

Administration

The active compound or pharmaceutical composition comprising the activecompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g. by ingestion);topical (including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g. by inhalation or insufflation therapyusing, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot, for example,subcutaneously or intramuscularly.

The subject may be a eukaryote, an animal, a vertebrate animal, amammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan,gibbon), or a human.

Formulations

While it is possible for the active compound to be administered alone,it is preferable to present it as a pharmaceutical composition (e.g.formulation) comprising at least one active compound, as defined above,together with one or more pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, stabilisers,preservatives, lubricants, or other materials well known to thoseskilled in the art and optionally other therapeutic or prophylacticagents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, buffers, adjuvants, stabilisers, or other materials, asdescribed herein.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g. human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activecompound with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations may be in the form of liquids, solutions, suspensions,emulsions, elixirs, syrups, tablets, losenges, granules, powders,capsules, cachets, pills, ampoules, suppositories, pessaries, ointments,gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses,electuaries, or aerosols.

Formulations suitable for oral administration (e.g. by ingestion) may bepresented as discrete units such as capsules, cachets or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or suspension in an aqueous or non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g. povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g. lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g. magnesium stearate, talc, silica);disintegrants (e.g. sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g. sodium lauryl sulfate); andpreservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid). Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activecompound therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal,intranasal, ocular, buccal, and sublingual) may be formulated as anointment, cream, suspension, lotion, powder, solution, past, gel, spray,aerosol, or oil. Alternatively, a formulation may comprise a patch or adressing such as a bandage or adhesive plaster impregnated with activecompounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth includelosenges comprising the active compound in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activecompound in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active compound in a suitableliquid carrier.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active compound is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include thosepresented as an aerosol spray from a pressurised pack, with the use of asuitable propellant, such as dichlorodifluoromethane,trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, orother suitable gases.

Formulations suitable for topical administration via the skin includeointments, creams, and emulsions. When formulated in an ointment, theactive compound may optionally be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the active compounds may beformulated in a cream with an oil-in-water cream base. If desired, theaqueous phase of the cream base may include, for example, at least about30% w/w of a polyhydric alcohol, i.e., an alcohol having two or morehydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol,sorbitol, glycerol and polyethylene glycol and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active compound through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionallycomprise merely an emulsifier (otherwise known as an emulgent), or itmay comprises a mixture of at least one emulsifier with a fat or an oilor with both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required.

Alternatively, high melting point lipids such as white soft paraffinand/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active compound, such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration (e.g. by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer's Solution,or Lactated Ringer's Injection. Typically, the concentration of theactive compound in the solution is from about 1 ng/ml to about 10 μg/ml,for example from about 10 ng/ml to about 1 μg/ml. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets. Formulations may be in the form ofliposomes or other microparticulate systems which are designed to targetthe active compound to blood components or one or more organs.

Dosage

It will be appreciated that appropriate dosages of the active compounds,and compositions comprising the active compounds, can vary from patientto patient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, and the age, sex, weight, condition,general health, and prior medical history of the patient. The amount ofcompound and route of administration will ultimately be at thediscretion of the physician, although generally the dosage will be toachieve local concentrations at the site of action which achieve thedesired effect without causing substantial harmful or deleteriousside-effects.

Administration in vivo can be effected in one dose, continuously orintermittently (e.g. in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician.

In general, a suitable dose of the active compound is in the range ofabout 100 μg to about 250 mg per kilogram body weight of the subject perday. Where the active compound is a salt, an ester, prodrug, or thelike, the amount administered is calculated on the basis of the parentcompound and so the actual weight to be used is increasedproportionately.

EXAMPLES

The following are examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Where molecular weight (Mw) is quoted as confirmation that the desiredcompound has been synthesised, this is the molecular weight of theprotonated compound detected using LC-MS, and is therefore one unithigher than the actual Mw of the compound, i.e. Mw+1.

Synthesis Details Route 1 Compound 1 (a)3-phenyl-3-hydroxy-dithioacrylic acid

Bright orange solid (5.9 g, 75%) from (4.67 ml, 40 mmol) ofacetophenone; FT-IR (ATR/cm⁻¹) 3055, 1542, 1450, 1234, 1059, 909, 751,674; ¹H NMR (CDCl₃) δ=7.30 (1H, s); 7.35-8.05 (5H, m), 15.18 (1H, s)

(b) Ethyl 3-phenyl-3-hydroxy-dithioacrylate

Brown oil (3.84 g, 68%) from (4.91 g, 25 mmol) of3-phenyl-3-hydroxy-dithioacrylic acid; FT-IR (ATR/cm⁻¹): 3062, 2970,2923, 2550, 1395, 1225, 1042, 948, 755; ¹H NMR (CDCl₃) δ=1.31 (3H, t),3.20 (2H, q), 6.84 (1H, s), 7.34-7.83 (5H, m), 15.06 (1H, s)

(c) 1-Phenyl-3-morpholin-4-yl-3-thioxo-propan-1-one

White crystalline solid (3.26 g, 80%) from (3.64 g, 16.25 mmol) of ethyl3-phenyl-3-hydroxy-dithioacrylate; FT-IR (ATR/cm⁻¹): 3023, 2908, 2871,1681, 1496, 1433, 1311, 1169, 1103, 953, 748; ¹H NMR (CDCl₃) δ=3.59-3.80(6H, m); 4.33 (2H, m); 4.72 (2H, s); 7.38-7.96 (5H, m)

(d) 1-phenyl-3-ethylsulfanyl-3-morpholin-4-yl-propenone

Brown oil (3.21 g, 97%)

(e) 4-morpholin-4-yl-6-(phenyl)-pyran-2-one (Compound 1)

White solid (0.38 g, 15%); mp 161-162° C.; FT-IR (ATR/cm⁻¹): 3049, 2956,2901, 2862, 1977, 1628, 1537, 1436, 1109, 761, 687; ¹H NMR (DMSO) δ=3.63(4H, t, 4.5 Hz, CH₂N), 3.81 (4H, t, 4.5 Hz, CH₂O), 5.37 (1H, d, 2 Hz,H-3), 7.17 (1H, d, 2 Hz, H-5), 7.61-7.65 (3H, m, ArH), 8.03-8.08 (2H, m,ArH); UV: λ_(max) (MeOH/nm): 291, 250; MS: m/z (LC-MS/ESP+): 258 (M⁺+1),211, 179, 133; Calcd C₁₅H₁₅NO₃. 0.1 EtOAc: C 69.51; H, 5.98; N, 5.26.Found: C, 69.54; H, 5.93; N, 4.96.

4-morpholin-4-yl-6-(4-(t-butyl)phenyl)-pyran-2-one (Compound 2)

White needles (0.61 g, 19%); mp 230-232° C.; FT-IR (ATR/cm⁻¹): 3109,3051, 2947, 2862, 1674, 1633, 1511, 1446, 1114, 941, 826, 782; ¹H NMR(DMSO) δ=1.42 (9H, s, (CH₃)₃C), 3.62 (4H, m, CH₂N), 3.79 (4H, m, CH₂O),5.34 (1H, bs, H-3), 7.10 (1H, bs, H-5), 7.63 (2H, d, 8.5 Hz, ArH), 7.96(2H, d, 8.5 Hz, ArH); UV: λ_(max)(MeOH/nm): 265.5, 235.5; MS: m/z(LC-MS/ESP+): 314 (M⁺+1); Calcd C₁₉H₂₃NO₃.0.1H₂O: C 72.40; H, 7.42; N,4.44. Found: C, 72.50; H, 7.48; N, 4.18.

4-morpholin-4-yl-6-(4-methoxyphenyl)-pyran-2-one (Compound 3)

White needles (0.43 g, 15%); mp 212-213° C.; FT-IR (ATR/cm⁻¹): 2969,2926, 1681, 1619, 1505, 1442, 1240, 1180, 1113, 789; ¹H NMR (DMSO)δ=3.60 (4H, t, 4.5 Hz, CH₂N); 3.79 (4H, t, 4.5 Hz, CH₂O); 3.94 (3H, s,MeO); 5.30 (1H, d, 2 Hz, H-3); 7.02 (1H, d, 2 Hz, H-5); 7.16 (2H, d, 9Hz, ArH); 7.98 (2H, d, 9 Hz, ArH); UV: λ_(max) (MeOH/nm): 226, 256,301.5; MS: m/z (LC-MS/ESP+): 288 (M⁺+1), 157; Calcd C₁₆H₁₇NO₄: C 66.89;H, 5.96; N, 4.88. Found: C, 66.65; H, 6.03; N, 4.51.

4-morpholin-4-yl-6-(4-chlorophenyl)-pyran-2-one (Compound 4)

White needles (0.31 g, 21%) from (1.55 g, 5 mmol) of1-(4-chloro-phenyl)-3-ethylsulfanyl-3-morpholin-4-yl-propenone; mp236-237° C.; FT-IR (ATR/cm⁻¹): 3040, 2969, 1681, 1624, 1535, 1235, 941,785; ¹H NMR (DMSO) δ=3.65 (4H, m, CH₂N); 3.83 (4H, m, CH₂O); 5.40 (1H,m, H-3); 7.22 (1H, m, H-5); 7.75 (2H, m, ArH); 8.09 (2H, m, ArH); UV:λ_(max) (MeOH/nm): 296.5, 254; MS: m/z (LC-MS/ESP+): 292-294 (M⁺+1);Calcd C₁₅H₁₄ClNO₃: C 61.76; H, 4.84; N, 4.80. Found: C, 61.55; H, 4.91;N, 4.55.

Route 2-Step (c) 2-Morpholin-1-yl-pyrimido[2,1-a]isoquinolin-4-one(Compound 5)

Prepared from 2-chloro-pyrimido[2,1-a]isoquinolin-4-one (0.230 g, 1mmol) and morpholine (0.35 ml, 4 mmol) to give white crystals (0.236 g,0.83 mmol, 83% yield). FT-IR (KBr disc): cm⁻¹ 3070, 2983, 2945, 2911,2864, 1701, 1641, 1574, 1546, 1522, 1488, 1427, 1402, 1286, 1225, 1116,773. m/z (EI): 281 (M⁺), 250, 224, 195, 168, 128, 101, 77. ¹H NMR 200MHz, DMSO): 3.82 (8H, s, morpholine-H), 5.73 (1H, s, H-3); 7.37 (1H, d,8 Hz, ArH); 7.75 (1H, m, ArH); 7.77 (1H, d, 5 Hz, ArH); 7.91 (1H, d, 5Hz, ArH); 8.62 (1H, d, 7.5 Hz, ArH); 8.88 (1H, d, 7.5 Hz, ArH)

2-(Thiomorpholin-4-yl)pyrimido[2,1-a]isoquinolin-4-one (Compound 6)

Pale yellow crystals (0.255 g, 0.86 mmol, 86% yield). Mp=240-242 (C. UV(max=354.5, 335.5, 320, 280.5, 261.5, 232, 200 nm (Methanol). ¹H NMR(200 MHz, CDCl3) (2.66 (4H, m); 4.06 (4H, m); 5.62 (1H, s); 7.01 (1H,d); 7.62 (3H, m); 8.60 (1H, d); 8.75 (1H, m). ES-MS m/z=298 (M+1). Anal.Calcd for C₁₆H₁₅N₃OS: C, 64.62; H, 5.08; N, 14.13. Found: C, 64.22; H,4.86; N, 13.94.

2-(2,5-Dimethyl-piperidin-1-yl)pyrimido[2,1-a]isoquinolin-4-one(Compound 7)

White crystals (0.126 g, 0.41 mmol, 41% yield). mp 214-216° C.λ_(max)=356, 336, 322, 261.5, 231.5, 200 nm (Methanol). m/z (ES⁺): 308(M⁺+1), 179, 133. ¹H NMR (200 MHz, CDCl₃) δ0.89 (3H, s); 0.93 (3H, s);1.65 (4H, m); 4.42 (2H, s); 5.62 (1H, s); 6.96 (1H, d); 7.61 (3H, m);8.59 (1H, d); 8.76 (1H, m). Anal. Calcd. for C₁₉H₂₁N₃O. 0.2CH₃OH: C,73.49; H, 7.00; N, 13.39. Found: C, 73.92; H, 6.77; N, 13.56.

2-(4-Methyl-piperazin-1-ly)pyrimido[2,1-a]isoquinolin-4-one (Compound 8)

White solid (0.095 g, 0.32 mmol, 32% yield. mp=Sublimes above 285° C.m/z (ES⁺) 295 (MH⁺), 257, 179. ¹H NMR (200 MHz, d₆-DMSO) 62.91 (3H, s);3.44 (8H, m); 5.95 (1H, s); 7.49 (1H, d); 7.95 (1H, m); 8.02 (2H, m);8.67 (1H, d); 8.99 (1H, m).

2-(3-Hydroxymethyl-piperidin-1-yl)pyrimido[2,1-a]isoquinolin-4-one(Compound 9)

White solid (0.157 g, 0.50 mmol, 50% yield). mp 165-166° C. ESMS m/z(ES⁺) 310 (M+H), 257, 179. ¹H NMR (200 MHz, CDCl₃) 1.75 (5H, m); 2.39(1H, m); 3.31 (1H, m); 3.59 (3H, m); 4.09 (2H, m); 5.64 (1H, s); 7.01(1H, d); 7.63 (3H, m); 8.63 (1H, d); 8.77 (1H, m).

2-[(Tetrahydro-furan-2-ylmethyl)-amino]pyrimido[2,1-a]isoquinolin-4-one(Compound 10)

White solid (0.173 g, 0.58 mmol, 58% yield). mp 174-175° C. ESMS m/z=296(M+H), 257, 179. ¹H NMR (200 MHz, CDCl₃) δ1.81 (4H, m); 3.56 (2H, d);3.73 (1H, q); 3.86 (1H, q); 4.07 (1H, m); 5.29 (1H, s, NH); 5.43 (1H,s); 6.96 (1H, d); 7.59 (3H, m); 8.57 (1H, d); 8.76 (1H, d)

2-[Bis-(2-hydroxy-ethyl)-amino]pyrimido[2,1-a]isoquinolin-4-one(Compound 11)

White solid (0.076 g, 0.26 mmol, 26% yield). mp 211-212° C. ESMS m/z=300(M+1), 257, 179. ¹H NMR (200 MHz, D₆DMSO) 63.90 (4H, m,); 5.62 (1H, s);7.38 (1H, d); 7.81 (1H, m); 7.94 (1H, d); 8.58 (1H, d); 8.85 (1H, d).Anal. Calcd for C₁₆H₁₇N₃O₃: C, 69.88; H, 6.19; N, 13.55. Found: C,69.70; H, 6.27; N, 13.44

2-(3-Hydroxy-pyrrolidin-1-yl)pyrimido[2,1-a]isoquinolin-4-one (Compound12)

Beige solid (0.211 g, 0.75 mmol, 75% yield). mp 240-241° C. UVλ_(max)=248.5, 258.0, 273.5, 344.5, 362.0 nm (Methanol). ESMS m/z=282(M+1), 257, 179, 133. ¹H NMR (200 MHz, d₆DMSO) 62.18 (2H, m); 3.45 (2H,m); 3.86 (2H, m); 4.52 (1H, m); 5.17 (1H, s); 7.36 (1H, d); 7.80 (1H,m); 7.94 (2H, d); 8.63 (1H, d); 8.86 (1H, d)

2-(Cis-2,6-dimethylmorpholin-4yl)pyrimido[2,1-a]isoquinolin-4-one(Compound 13)

White crystals (0.088 g, 0.28 mmol, 56% yield). mp 208-209° C. ESMSm/z=310 (M+1), 257, 179, 101. ¹H NMR (200 MHz, CDCl₃) δ1.29 (6H, d);2.68 (2H, dd); 3.70 (2H, m); 4.30 (2H, m); 5.63 (1H, s); 7.06 (1H, d);7.67 (3H, m); 8.65 (1H, d); 8.81 (1H, d)

2-[Benzyl-(2-hydroxy-ethyl)-amino]pyrimido[2,1-a]isoquinolin-4-one(Compound 14)

White crystals (0.077 g, 0.22 mmol, 44% yield). ESMS m/z=346 (M+1), 257,179, 101. ¹H NMR (200 MHz, d₆DMSO) δ 3.77 (4H, m); 4.97 (2H, m); 5.63(1H, s); 7.41 (6H, m); 7.95 (3H, m); 8.63 (1H, d); 8.84 (1H, d).

2[(2-hydroxy-ethyl)-methyl-amino]-pyrimido[2,1-a]isoquinolin-4-one(Compound 15)

White crystals (0.079 g, 0.29 mmol, 58% yield). ESMS m/z 270 (M+1), 257,179, 133, 101. ¹H NMR (200 MHz, CDCl₃) δ 3.15 (3H, s); 3.92 (4H, m);5.55 (1H, s); 7.00 (1H, d); 7.64 (3H, m); 8.62 (1H, d); 8.73 (1H, d).

2-[(2-Hydroxy-2-phenyl-ethyl)-methyl-amino]-pyrimido[2,1-a]isoquinolin-4-one(Compound 16)

Off-white crystalline solid (0.115 g, 0.33 mmol, 66% yield). mp 195-196°C. UV λ_(max)=354, 334.5, 320, 259, 232, 200 nm (Methanol). ESMS m/z=346(M+1). ¹H NMR (200 MHz, CDCl₃) δ2.94 (3H, s); 3.99 (2H, m); 4.60 (1H,s); 5.13 (1H, m); 5.54 (1H, s); 7.04 (1H, d); 7.36 (5H, m); 7.71 (3H,m); 8.65 (1H, d); 8.82 (1H, m). Anal Calcd for C₂₁H₁₉N₃O₂.0.15CH₃OH: C,72.48; H, 5.65; N, 11.98. Found: C, 72.57; H, 5.51; N, 11.89

3-[Methyl-(4-oxo-4H-pyrimido[2,1-a]isoquinolin-2-yl)-amino]-propionitrile(Compound 17)

Off-white crystalline solid (0.067 g, 0.24 mmol, 48% yield). mp 166-167°C. UV λ_(max)=352, 334, 316, 200 nm (Methanol). ESMS m/z=279 (M+1). ¹HNMR (200 MHz, CDCl₃) δ2.80 (2H, t); 3.18 (3H, s); 4.08 (2H, t); 5.58(1H, s); 7.01 (1H, d); 7.71 (3H, m); 8.68 (1H, d); 8.76 (1H, m). AnalCalcd for C₁₆H₁₄N₄O: C, 69.05; H, 5.07; N, 20.13. Found: C, 68.47; H,4.99; N, 19.93.

2-(2-Thiophen-2-yl-ethylamino)-pyrimido[2,1-a]isoquinolin-4-one(Compound 18)

Off-white crystalline solid (0.115 g, 0.36 mmol, 72% yield). mp 162-163°C. UV λ_(max)=352, 334, 318.5, 253, 229.5, 200 nm (Methanol). ESMSm/z=322 (M+1), 301, 181. ¹H NMR (200 MHz, CDCl₃) δ3.15 (3H, m); 3.56(2H, m); 5.07 (1H, s); 5.47 (1H, s); 6.73 (2H, m); 7.16 (2H, m); 7.61(3H, m); 8.60 (1H, d); 8.75 (1H, m). Anal. Calcd. for C₁₈H₁₅N₃OS: C,67.27; H, 4.70; N, 13.07. Found: C, 66.84; H, 4.57; N, 13.07.

2-(2,3-Dihydroxypropylamino)-pyrimido[2,1-a]isoquinolin-4-one (Compound19)

Off-white crystalline solid (0.045 g, 0.16 mmol, 32% yield). mp 215-216°C. ESMS m/z=286 (M+1), 157, 110. ¹H NMR (200 MHz, d₆-DMSO) δ1.14 (2H,m); 3.47 (2H, m); 3.78 (1H, m); 4.47 (1H, t); 4.77 (1H, t); 5.01 (1H,d); 5.51 (1H, s); 7.36 (1H, d); 7.80 (1H, m); 7.94 (2H, m); 8.63 (1H,d); 8.86 (1H, m)

2-(2-Hydroxypropylamino)-pyrimido[2,1-a]isoquinolin-4-one (Compound 20)

Off-white crystalline solid (0.072 g, 0.27 mmol, 54% yield). mp 199-200°C. ESMS m/z=270 (M+1), 179, 157, 133, 111. ¹H NMR (200 MHz, d₆-DMSO)δ1.23 (3H, d); 3.55 (2H, m); 3.95 (2H, t); 4.93 (1H, d); 5.50 (1H, s);7.36 (1H, d); 7.82 (1H, m); 7.95 (2H, m); 8.63 (1H, d); 8.88 (1H, m)

2-[2-Hydroxy-2-(3-hydroxy-phenyl)-ethylamino]-pyrimido[2,1-a]isoquinolin-4-one(Compound 21)

Off-white crystalline solid (0.117 g, 0.34 mmol, 68% yield). mp 159-161°C. UV λ_(max)=352.5, 333, 317, 257, 231, 200 nm (Methanol). ESMS m/z=348(M+1), 239, 222, 133. ¹H NMR (200 MHz, d₆-DMSO) δ4.81 (2H, m); 5.52 (1H,s); 5.64 (1H, d); 6.75 (1H, m); 6.96 (2H, m); 7.24 (1H, t); 7.39 (1H,d); 7.82 (1H, m); 7.96 (2H, m); 8.65 (1H, d); 8.89 (1H, m); 9.48 (1H, brs). Anal. Calcd. for C₂₀H₁₇N₃O₃.0.3CH₂Cl₂: C, 65.07; H, 4.65; N, 11.19.Found: C, 65.05; H, 4.92; N, 11.06.

2-(2-Hydroxy-ethylamino)-pyrimido[2,1-a]isoquinolin-4-one (Compound 22)

Off-white crystalline solid (0.091 g, 0.36 mmol, 72% yield). mp 218-221°C. UV λ_(max)=352, 333.5, 316, 226.5, 200 nm (Methanol). ESMS m/z=256(M+1), 229. ¹H NMR (200 MHz, CDCl₃) δ3.45 (2H, m); 3.71 (2H, m); 4.92(1H, t); 5.49 (1H, s); 7.39 (1H, d); 7.83 (1H, m); 7.96 (2H, m); 8.64(1H, d); 8.89 (1H, m). Anal. Calcd. for C₁₄H₁₃N₃O₂: C, 65.87; H, 5.13;N, 16.46. Found: C, 65.40; H, 4.96; N, 16.12.

Additional examples of compounds synthesised using synthetic route 2 aregiven in the table below.

Mw Compound Structure LC-MS 23

310 24

264 25

266.25

Route 3

Examples of compounds synthesised using synthetic route 3 and syntheticroute 4 are listed in the following table. An asterix on the structureindicates the place at which the substituent and core structure arejoined. So, for example, a core structure of

where

defines a compound with structure

Compound Mw No. R LC-MS Purity 26

372 85

27

346 85 28

376 85

The wavey bonds,

in the structures of compounds 27 and 28 indicate a bond pointing eitherup or down (axial or equatorial positions). The structures thereforerepresent a dimethylmorpholino group having a mixture of cis and transmethyl groups.

Route 4

Examples of compounds synthesised using synthetic route 4 are listed inthe tables below.

Com- Mw pound LC- Puri- No. Structure MS ty 29

284 90

Compound Mw No. R LC-MS Purity

30

283 95 31

417 95 32

364 95 33

330 85 34

392 90 35

342 95 36

334 95

37

286 90 38

364 95 39

316 90 40

300 85 41

342 90 42

417 85 43

283 90 44

342 95 45

302 85

46

364 95 47 CF₃ 326 85

48 H 273 85

49

373 95 50

407 90

Compound Mw No. R LC-MS Purity 51

266 90 52

260 90 53

262 90 54

261 90 55

346 90 56

330 90 57

343 90 58

322 85 59

300 90 60

316 90 61

248 90 62

292 90 63

314 85 64

309 85 65

297 95 66

264 95 67

264 85 68

314 95 69

308 95 70

336 85 71

298 95 72

314 95

Route 4a

Examples of compounds synthesised using synthetic route 4a are listed inthe following tables.

Com- Mw pound No. R₁ R₂ LC-MS Purity 73

H 339 85 74

H 341 95 75

H 437 95 76

H 371 95 77

H 399 95 78

H 343 95 79

H 397 90 80

H 355 90 81

H 343 95 82

H 357 95 83

H 385 95 84

381 95 85

H 435 95 86

H 481 95 87

481 95 88

H 459 95 89

H 391 95 90

H 421 95 91

H 409 95 92

H 419 95 93

H 405 95 94

H 481 95 95

H 409 95 96

H 454 95 97

H 475 90 98

H 527 90 99

H 459 95 100

H 477 95 101

H 451 85 102

H 406 95 103

H 405 95 104

H 406 90 105

H 434 90 106

462 90 107

H 420 90 108

H 505 95 109

H 389 95 110

H 405 95 111

H 383 90 112

H 354 90 113

H 359 90 114

H 399 90 115

H 413 95 116

H 375 95 117

H 400 90 118

H 414 90 119

462 90 120

Me 386 90 121

H 372 95 122

H 492 95 123

H 464 85 124

H 437 90 125

H 373 95 126

H 421 95 127

H 359 95 128

H 387 95 129

H 498 90 130

H 463 90 131

H 397 85 132

H 411 95 133

H 385 95 134

H 412 90 135

H 413 90 136

H 474 90 137

412 90 138

417 95 139

371 95 140

399 95 141

398 90 142

453 95 143

486 95 144

385 95

145 H H 316 85 146

386 90 147

414 95 148

399 90

Route 4b

Examples of compounds synthesised using synthetic route 4b are listed inthe following tables.

Compound Mw No. R₁ R₂ LC-MS Purity 149

H 339 90 150

H 399 90 151

H 439 90 152 Me H 315 85 153

H 341 85 154

H 437 90 155

H 371 90 156

H 397 90 157

H 385 90 158

H 371 90 159

H 343 90 160

H 394 90 161

H 355 90 162

H 481 90 163

H 405 90 164

H 436 90 165

H 451 90 166

481 90 167

H 435 90 168

H 421 90 169

H 409 90 170

H 419 90 171

H 405 90 172

H 481 90 173

H 454 90 174

H 421 90 175

H 475 90 176

H 527 90 177

H 451 90 178

H 459 90 179

H 477 90 180

H 419 90 181

H 434 85 182

H 406 90 183

H 405 90 184

H 435 85 185

449 90 186

H 375 90 187

H 504 90 188

H 405 90 189

H 340 90 190

H 375 90 191

H 427 90 192

H 419 90 193

H 399 90 194

H 415 90 195

H 508 90 196

H 449 90 197

H 415 85 198

H 463 90 199

H 495 90 200

H 449 90 201

H 373 90 202

H 421 90 203

H 492 90 204

H 385 90 205

H 474 90 206

H 410 90 207

H 412 90 208

H 412 90 209

H 503 90 210

H 397 90 211

H 381 90 212

H 394 90 213

H 406 85 214

H 392 TFA salt 90 215

441 90 216

453 90 217

477 90 218

417 90 219

367 90 220

387 90 221

385 90 222

371 90

Compound Mw No. R₁ R₂ LC-MS Purity 223

414 TFA salt 90

Route 4c(i)

Examples of compounds synthesised according to synthetic route 4c(i) arelisted in the following tables.

Compound Mw No. R₁ R₂ LC-MS Purity 224

H 397 90 225

H 326 90 226

H 361 90 227

H 401 90 228

H 445 90 229

H 413 90 230

H 391 90 231

H 403 90 232

H 491 85 233

H 383 90 234

H 397 90 235

341 90 236

427 90 237

439 90

Route 4c(ii)

Examples of compounds synthesised according to synthetic route 4c(ii)are listed in the following tables.

Compound Mw No. R₁ R₂ LC-MS Purity 238

H 357 85 239

H 383 85 240

H 383 85 241

H 371 85 242

H 445 85 243

H 453 85 244

H 437 85 245

H 445 85 246

Me 391 85 247

H 435 85 248

H 407 85 249

H 395 85 250

H 467 85 251

H 395 85 252

H 461 85 253

H 437 85 254

H 391 85 255

H 392 85 256

H 437 85 257

H 489 85 258

439 85 259

456 85 260

403 85 261

427 85 262

H 449 85 263

H 384 85

Route 4d(iii)

Examples of compounds synthesised according to synthetic method 4d(iii)(from a precusor synthesised according to route 4d(ii)) are listed inthe following tables.

Compound Mw No. R LC-MS Purity 265

373.03 90 266

401.09 90 267

387.06 90 268

345 90 269

387.06 90 270

423 90 271

397 90 272

367 90 273

441.54 90 274

421.12 90 275

441.54 90 276

422 90 277

449.13 85 278

461.07 85 279

466.09 90

Mw Compound No. R LC-MS Purity 280

420 90

Mw Compound No. R LC-MS Purity 281

480 90

Route 4d(iv)

Examples of compounds made according to synthetic route 4d(iv) (from aprecursor synthesised according to route 4d(i)) are listed in the tablesbelow.

Mw Compound No. R₁ R₂ LC-MS Purity 282

H 435 85

Route 4d(v)

Examples of compounds synthesised according to route 4d(v) (from aprecursor synthesised according to route 4d(i)) are listed in the tablesbelow.

Mw Compound No. R LC-MS Purity 264

436 85

Route 5 Compound 283 (a) Methyl3(4-chlorophenyl)-3-oxo-triphenylphosphanyl-propionate

Purification by column chromatography (ethyl acetate:petroleum ether40-60°, 3:2) yielded to a white solid (3.41 g, 77.2%); mp 136° C.; IR(KBr/cm⁻¹): 3074, 3056, 2940, 1662, 1314, 1247, 1105, 1077, 752, 695; ¹HNMR (CDCl₃) δ=3.07 (3H, s), 7.18-7.72 (19H, m); hMS m/z (EI): 472.0993(M⁺, Calcd 472.0995 for C₂₈H₂₂O₃PCl), 472, 361, 277, 201, 163.

(b) Methyl 3-(4-chlorophenyl) propiolate

Purification by column chromatography (ethyl acetate:petroleum ether40-60°, 15:85) yielded to a white solid (1.17 g, 88.5%); mp 90° C.; ¹HNMR (CDCl₃) δ=3.77 (s, 3H), 7.27-7.65 (4H, m); IR (KBr/cm⁻¹): 3049,3035, 2964, 2226, 1718, 1489, 1293, 1170, 823, 721; HRMS m/z (EI):194.0130 (M⁺, Calcd 194.0135 for C₁₀H₇O₂Cl), 194, 163, 136, 99, 74.

(c) 4-[(4-chlorophenyl)-2-oxo-3-butynyl) carbonyl]morpholine lithiumsalt

yielded to a white solid (0.42 g, 50%); mp>320° C.; ¹H NMR (d⁶-DMSO)δ=3.46 (4H, m), 3.61 (4H, m), 5.06 (1H, m), 7.56 (4H, s); IR (KBr/cm⁻¹):3407, 3091, 2961, 2200, 1571, 1506, 1230, 1116, 961, 755; HRMS m/z (EI):291.0445 (M⁺, Calcd 291.0662 C₁₅H₁₄ClNO₃), 291, 263, 163, 136, 86.

(d) 6-(4-chlorophenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 284)

Purification by column chromatography (ethyl acetate:petroleum ether40-60°, 15:85) yielded to a white solid (0.15 g, 50%); mp 250° C.; IR(KBr/cm⁻¹): 3071, 2965, 1643, 1559, 1410, 1124, 899, 854; ¹H NMR (CDCl₃)δ=3.41 (4H, t), 3.83 (4H, t), 5.45 (1H, d), 6.51 (1H, d), 7.42 (2H, d);7.58 (2H, d); HRMS m/z (EI): 291.0666 (M⁺, Calcd 291.0662 forC₁₅H₁₄O₃NCl), 291-293, 263-265, 205-207, 136-138; UV:λ_(max)(MeOH)=354.0 nm; Anal. Calcd for C₁₅H₁₄O₃NCl. 0.1H₂O: C, 61.38;H, 4.88; N, 4.77; Cl, 12.08. Found: C, 61.58; H, 4.99; N, 4.34; Cl,12.40.

2-(4-Morpholinyl)-6-phenyl-4H-pyran-4-one (Compound 285)

Pale green solid (0.38 g, 63%); mp 148-150° C.; IR (KBr/cm⁻¹): 1648,1561, 1230, 1108, 1030, 896, 775. ¹H NMR: δ (d₆-DMSO): 3.55 (4H, m,CH₂N), 3.85 (4H, m, CH₂O), 5.54 (1H, d), 6.75 (1H, d), 7.63 (3H, m,Ar—H), 8.00 (2H, m, Ar—H); HRMS m/z (EI): 257.1047 (M⁺, Calcd 257.1052for C₁₅H₁₅NO₃), 257, 229, 200, 171, 131, 111, 102, 86, 77; Anal. Calcdfor C₁₅H₁₅NO₃H₂O: C, 68.53; H, 5.71; N, 5.33. Found: C, 68.53; H, 5.90;N, 5.14.

6-(2-methoxyphenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 286)

White solid (0.297 g, 23%); mp 125-127° C.; IR (KBr/cm⁻¹): 3077, 3001,2968, 1641, 1604, 1562, 1404, 1241, 1121, 1019, 862, 761; ¹H NMR (CDCl₃)δ=3.34 (4H, t), 3.75 (4H, t), 3.83 (3H, s), 5.39 (1H, d), 6.72 (1H, d),6.93-7.01 (2H, m), 7.33-7.52 (2H, m); HRMS m/z (EI): 287.1171 (M⁺, Calcd287.1158 for C₁₆H₁₇O₄N), 287, 259, 244, 131, 111; UV: λ_(max)(MeOH)=358nm; Anal. Calcd for C₁₆H₁₇O₄N. 0.2H₂O: C, 66.06; H, 6.03; N, 4.81.Found: C, 66.13; H, 5.90; N, 4.73.

6-(3-methoxyphenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 287)

White solid (1.33 g, 99%); mp 115-117° C.; IR (KBr/cm⁻¹): 3078, 2975,1647, 1536, 1420, 1239, 1123, 879, 793; ¹H NMR (CDCl₃) δ=3.41 (4H, t),3.84 (7H, m), 5.45 (1H, d), 6.53 (1H, d), 6.97-7.41 (4H, m); HRMS m/z(EI): 287.1154 (M⁺, Calcd 287.1158 for C₁₆H₁₇O₄N), 287, 259, 200, 173,135, 102; UV: λ_(max)(MeOH)=356 nm; Anal. Calcd for C₁₆H₁₇O₄N: C, 66.67;H, 5.93; N, 4.62. Found: C, 66.9; H, 5.93; N, 4.62.

6-(4-methoxyphenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 288)

White solid (1.29 g, 96%); mp 220° C.; IR (KBr/cm⁻¹): 3085, 2968, 1649,1600, 1513, 1405, 1259, 1190, 835; ¹H NMR (CDCl₃) δ=3.40 (4H, t), 3.82(4H, t), 3.84 (3H, s), 5.41 (1H, d), 6.43 (1H, d), 6.95 (d, 2H), 7.59(d, 2H); HRMS m/z (EI): 287.1158 (M⁺, Calcd 287.1158 for C₁₆H₁₇O₄N),287, 287, 259, 201, 132; UV: λ_(max)(MeOH)=358 nm; Anal. Calcd forC₁₆H₁₇O₄N: C, 66.67; H, 5.93; N, 4.62. Found: C, 66.58; H, 5.90; N,4.84.

6-(4-tert-butylphenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 289)

White solid (0.94 g, 75.5%); mp 156° C.; IR (KBr/cm⁻¹): 3071, 3058,2960, 1648, 1571, 1404, 1362, 1121, 900, 826; ¹H NMR (CDCl₃) δ=1.28 (9H,s), 3.37 (4H, t), 3.78 (4H, t), 5.39 (1H, d), 6.47 (1H, d), 7.42 (2H,d), 7.54 (2H, d); HRMS m/z (EI): 313.1684 (M⁺, Calcd 313.1678 forC₁₉H₂₃O₃N), 313, 285, 270, 256, 213, 143; UV: λ_(max)(MeOH)=358 nm;Anal. Calcd for C₁₉H₂₃O₃N. 0.2H₂O: C, 71.99; H, 7.44; N, 4.42. Found: C,72.14; H, 7.35; N, 4.44.

6-(2-fluorophenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 290)

White solid (0.67 g, 76%); mp 137-138° C.; IR: (KBr)/(cm⁻¹): 3059, 3028,2928, 1640, 1570, 1405, 1119, 756; ¹H NMR (CDCl₃) δ=3.38 (4H, t), 3.76(4H, t), 5.40 (1H, d), 6.54 (1H, d), 7.07-7.23 (2H, m), 7.34-7.57 (2H,m); HRMS (EI) m/z 275.0950 [M⁺ calcd 275.0958 for C₁₅H₁₄O₃NF], 275, 247,189, 161, 134, 120, 86; UV: λ_(max)(MeOH)=244.5 nm; Anal. Calcd forC₁₀H₇O₂F. 0.2CH₂Cl₂: C, 64.6; H, 5.2; N, 5.0. Found: C, 64.8; H, 5.0; N,4.9.

6-(3-fluorophenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 291)

White solid (0.10 g, 11%); mp 169-170° C.; IR: (KBr)/(cm⁻¹): 3055, 2929,1650, 1564, 1403, 1245, 1114, 877; ¹H NMR (CDCl₃) δ=3.39 (4H, t), 3.76(4H, t), 5.40 (1H, d), 6.49 (1H, d), 7.08-7.45 (4H, m); HRMS (EI) m/z275.0946 [M⁺ calcd 275.0958 for C₁₅H₁₄O₃NF], 275, 247, 189, 161, 120,95; UV: λ_(max)(MeOH)=247 nm; Anal. Calcd for C₁₀H₇O₂F. 0.5 CH₂Cl₂: C,58.6; H, 4.8; N, 4.4. Found: C, 58.8; H, 4.6; N, 4.3.

6-(4-fluorophenyl)-2-(4-morpholinyl)-4H-pyran-4-one (Compound 292)

White solid (0.319 g, 82%); mp 216-217° C.; IR (KBr/cm⁻¹): 3065, 3010,2969, 2910, 1641, 1560, 1411, 1239, 1123, 856, 784; ¹H NMR (CDCl₃)δ=3.36 (4H, t), 3.78 (4H, t), 5.39 (1H, d), 6.43 (1H, d), 7.04-7.16 (2H,m), 7.55-7.65 (2H, m); HRMS (EI) m/z 275.0946 [M⁺ calcd 275.0958 forC₁₅H₁₄O₃NF], 275, 247, 210, 182, 120, 86; UV: max(MeOH)=247 nm; Calcdfor C₁₀H₇O₂F. 0.3CH₂Cl₂: C, 61.1; H, 4.9; N, 4.7. Found: C, 61.4; H,4.4; N, 4.7.

Route 6 Compound 293 (a) Methyl 1-hydroxy-2-naphthoate

Prepared from 1-hydroxy-2-naphthoic acid (9.4 g, 50 mmol), affording2.85 g (14 mmol, 28% yield) as an off white solid: mp 78-79° C. IR(KBr): 3051; 2953; 1662; 1635; 1438; 1336; 772 cm⁻¹. ¹H NMR (200 MHz.,CDCl₃) δ 3.91 (3H, s); 7.19 (1H, d, J=9 Hz.); 7.48 (2H, m); 7.68 (2H, d,J=9 Hz.); 8.33 (1H, d, J=8 Hz.); 11.88 (1H, s). EIMS m/z=202 (M+); 170;114.

(b) 1-(1-Hydroxynaphth-2-yl)-3-(morpholin-4-yl)-propan-1,3-dione

Prepared from methyl 1-hydroxy-2-naphthoate (2.28 g, 11.3 mmol),affording 2.49 g (8.3 mmol, 74% yield) of the title compound as anoff-white powder. mp 128-130° C. IR (KBr) 1658; 1620; 1223; 1114; 804cm⁻¹. ¹H NMR (200 MHz., d₆-DMSO) δ 3.61 (4H, m); 3.72 (4H, m); 4.50 (2H,s); 7.49 (1H, d, 8.9 Hz.); 7.72 (1H, dt, J=1.2 Hz., 7.5 Hz.); 7.85 (1H,dt, J=1.2 Hz., 8.2 Hz.); 7.92 (1H, d, J=8.9 Hz.); 8.03 (1H, d, 8.0 Hz.);8.46 (1H, d, 8.2 Hz.); 13.72 (1H, bs). EIMS m/z=299 (M+); 212; 170; 87.

(c) 7,8-Benzo-2-(morpholin-4-yl)-chromen-4-one (Compound 293)

Prepared from1-(1-hydroxynaphth-2-yl)-3-(morpholin-4-yl)-propan-1,3-dione (2.4 g, 8.0mmol), affording 1.43 g (5.1 mmol, 63% yield) of the desired compound aswhite crystals. mp 267-269° C. IR (KBr) 1641; 1626; 1605; 1509; 1562;1420; 1240; 117; 920 cm⁻¹. ¹H NMR (200 MHz., d₆-DMSO) δ 3.74 (4H, m);3.91 (4H, m); 5.79 (2H, s); 7.88 (1H, d, 8.9 Hz.); 8.02 (2H, m); 8.16(1H, m), 8.56 (1H, m). EIMS m/z=281 (M+); 224; 196; 170.

8-Phenyl-2-(morpholin-4-yl)-chromen-4-one (Compound 294)

Off-white powder (0.770 g, 2.51 mmol, 74% yield): mp 183-185° C. IR(KBr): 3419; 1621; 1563; 1414; 1252; 1119; 990; 755; 700 cm⁻¹. ¹H NMR(200 MHz., d₆-DMSO) δ 3.45 (4H, m, morpholine); 3.74 (4H, m,morpholine); 5.66 (1H, s, chromenone 3-H); 7.57 (4H, m); 7.73 (3H, m);8.06 (1H, m). ¹³C NMR (50 MHz., d⁶-DMSO) δ 44.8; 65.5; 86.4; 123.5;124.4; 125.1; 128.4; 128.8; 129.7; 130.2; 133.5; 136.0; 150.4; 162.5;175.4. EIMS m/z=307 (M+); 292; 250; 222; 196; 168; 139. Anal. Calcd forC₁₉H₁₇NO₃.0.2H₂O: C, 73.39; H, 5.64; N, 4.50. Found: C, 73.36; H, 5.21;N, 4.22.

2-piperidin-1-yl-benzo[h]chromen-4-one (Compound 295)

Pale brown solid. (0.034 g, 0.12 mmol, 32% yield) mp 205-207° C. ¹H NMR(200 MHz, d₆-DMSO) δ 1.69 (6H, s); 3.56 (4H, s); 5.59 (1H, s); 7.55 (2H,m); 7.83 (2H, q); 8.21 (1H, d); 8.24 (1H, m). EIMS m/z=279 (M⁺), 224,170, 127, 114, 87. Anal. Calcd for C₁₈H₁₇NO₂. 0.1 CH₂Cl₂: C, 75.53; H,6.02; N, 4.87. Found: C, 75.81; H, 5.80; N, 4.82.

2-(Thiomorpholin-4-yl)-benzo[h]chromen-4-one (Compound 296)

Orange solid. (0.39 g, 1.31 mmol, 46% yield), mp 171-173° C. FT-IR 3087,2963, 1642, 1604, 1562 cm⁻¹. ¹H NMR (200 MHz, d₆-DMSO) δ 2.86 (4H, m);4.06 (4H, m); 5.80 (1H, s); 7.84 (2H, m); 8.00 (2H, q); 8.12 (1H, m);8.46 (1H, m); EIMS m/z=297 (M⁺), 224, 170, 127, 114, 87. Anal. Calcd forC₁₇H₁₅NO₂S 0.3 CH₃COOC₂H₅: C, 67.39; H, 5.45; N, 4.29. Found: C, 67.22;H, 5.14; N, 4.14.

Compound 297 Synthesis of Starting Material5,6,7,8-Tetrahydro-1-hydroxy-2-naphthoic acid

A mixture of 5,6,7,8-tetrahydro-1-naphthol (7.42 g, 50 mmol) andpotassium carbonate (25.5 g, 185 mmol) were placed in a glass tubeinside a stainless steel pressure reactor. The reactor was charged withCO₂ at 40 bar and then heated to 145° C. The pressure rose to 60 bar andthen slowly dropped to 20 bar over the 3 day reaction period. The bombwas cooled and the solid product was taken up in water (˜500 ml) andacetone (˜500 ml). The mixture was evaporated in vacuo to remove theacetone and then washed with DCM (3×150 ml). The aqueous was acidifiedwith 2M hydrochloric acid to give a white suspension. This was extractedwith DCM (4×250 ml), which was then dried over sodium sulphate andevaporated in vacuo to give the crude product. This was recrystallisedfrom aqueous ethanol and dried under high vacuum to provide 8.64 g (45mmol, 90% yield) of the title compound as a pale brown powder. ¹H NMR(200 MHz., d⁶-DMSO) δ 1.81 (4H, m); 2.67 (2H, m); 2.81 (2H, m); 6.73(1H, d); 7.61 (1H, d); 11.78 (1H, bs). EIMS m/z=192 (M+); 174; 146

7,8,9,10-Tetrahydrobenzo[h]-2-(morpholin-4-yl)-chromen-4-one (Compound297)

Off-white powder: mp 220-222° C. IR (KBr): 1628; 1592; 1561; 1246; 1116;790 cm⁻¹. ¹H NMR (200 MHz., d₆-DMSO) δ 1.87 (4H, m); 2.90 (4H, m); 3.59(4H, m); 3.82 (4H, m); 5.56 (1H, s); 7.17 (1H, d); 7.72 (1H, d). EIMSm/z=285 (M+); 270; 228; 200; 175; 146. Anal. Calcd for C₁₇H₁₉NO₃: C,71.56; H, 6.71; N, 4.91. Found: C, 71.49; H, 6.76; N, 4.83.

Compound 298 Alternative Step (a) for this Compound Methyl5-bromo-2-hydroxybenzoate

Prepared from 5-bromo-2-hydroxybenzoic acid (3.26 g, 15 mmol) accordingto general method A, affording 2.45 g (10.6 mmol, 71% yield) as anoff-white powder. ¹H NMR (200 MHz., CDCl₃) δ 3.89 (3H, s, CH₃); 6.81(1H, d, J=8.8 Hz., 3-H); 7.46 (1H, dd, J=8.8, 2.5 Hz., 4-H); 7.89 (1H,d, J=2.5 Hz., 6-H); 10.62 (1H, s, OH).

Methyl 2-hydroxy-5-phenylbenzoate

A solution of phenylboronic acid (1.34 g, 11.0 mmol) and methyl5-bromo-2-hydroxybenzoate (2.42 g, 10.5 mmol) in acetone (25 ml) wastreated with water (30 ml), followed by potassium carbonate (3.77 g,27.3 mmol) and finally, palladium (II) acetate (0.16 g, 0.7 mmol). Uponaddition of the palladium, the reaction mixture rapidly darkened. Thereaction mixture was heated to reflux and stirred for 6 h. After coolingthe dark mixture, ether (40 ml) was added, stirred vigorously anddecanted. This extraction process was repeated an additional four times.The ethereal extracts were dried over sodium sulphate and evaporated invacuo to give a yellow liquid. The crude product was dissolved in petroland loaded onto a silica flash column. The column was eluted withpetrol, followed by 5-10% ethyl acetate in petrol. The second productcollected was evaporated in vacuo and then recrystallised from petrol toprovide a white crystalline solid (1.35 g, 5.90 mmol, 56% yield). ¹H NMR(200 MHz., d⁶-DMSO) δ 4.05 (3H, s); 7.20 (1H, m); 7.50-7.58 (3H, m);7.74 (2H, m); 7.97 (1H, m); 8.13 (1H, m); 10.67 (1H, s, OH).

2-(Morpholin-4-yl)-6-phenylchromen-4-one (Compound 298)

Off-white powder: mp 218-220° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.66 (4H,m); 3.85 (4H, m); 5.68 (1H, s, 3-H); 7.57 (3H, m); 7.72 (1H, d, 8-H);7.83 (2H, m); 8.08 (1H, dd, 7-H); 8.24 (1H, d, 5-H). EIMS m/z=307 (M+);196; 168. IR (KBr): 1611; 1558; 1428; 1245; 1119; 768 cm⁻¹. Anal. Calcd.for C₁₉H₁₇NO₃.0.2H₂O: C, 73.39; H, 5.64; N, 4.50. Found: C, 73.41; H,5.45; N, 4.28.

7-(2,6-Dichlorobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound299)

Off-white powder. ¹H NMR (200 Mhz, d₆-DMSO) δ 3.62 (4H, m); 3.82 (4H,m); 5.44 (2H, s); 5.55 (1H, s); 7.13 (1H, dd, J=2.4, 8.8 Hz.); 7.43 (1H,d, J=2.4 Hz.); 7.57-7.73 (3H, m); 7.94 (1H, d, J=8.8 Hz.)

2-morpholin-4-yl-chromen-4-one (Compound 300)

White powder. mp 143° C. IR: (KBr)/(cm⁻¹): 3067, 3035, 2960, 1620, 1555,1410, 1252, 1122, 1068, 770. ¹H NMR: δ(d₆-DMSO): 3.19 (4H, t, J=4.5,CH₂N); 3.87 (4H, t, J=4.5, CH₂O); 5.67 (1H, s, H-4); 7.26 (2H, m, Ar—H);7.49 (2H, m, Ar—H). HRMS m/z (EI): 231.0890 (M⁺, Calcd 231.0895 forC₁₃H₁₃NO₃), 214, 202, 172, 145, 118, 101, 89, 77. Anal. Calcd forC₁₃H₁₃NO₃: C, 67.52; H, 5.67; N, 6.06. Found: C, 67.28; H, 5.43; N,5.81.

2-morpholin-benzo<g>-chromen-4-one (Compound 301)

Pale brown solid. mp 219° C. IR (KBr)/(cm⁻¹): 3048, 2906, 2869, 1598,1569, 1464, 1424, 1356, 1252, 1118, 791. ¹H NMR: δ(d₆-DMSO): 3.60 (4H,t, J=4.5, CH₂N); 3.88 (4H, t, J=4.5, CH₂O); 5.54 (1H, s, H-4); 7.55 (1H,m, Ar—H); 7.74 (1H, m, Ar—H); 8.04 (1H, m, Ar—H); 8.74 (1H, m, Ar—H).HRMS m/z (EI): 281.1038 (M⁺, Calcd 281.1052 for C₁₇H₁₅NO₃), 224, 196,170, 142, 127, 114, 98. Anal. Calcd for C₁₇H₁₅NO₃.0.25H₂O: C, 71.43; H,5.25; N, 4.90. Found: C, 71.37; H, 5.04; N, 4.85.

8-Methyl-2-morpholin-4-yl-chromen-4-one (Compound 302)

Orange solid. mp 148° C. IR: (KBr)/(cm⁻¹): 3069, 2963, 2860, 1629, 1570,1411, 1251, 1118, 778. ¹H NMR: δ(d₆-DMSO): 2.51 (3H, s, Me); 3.60 (4H,t, J=5, CH₂N); 3.85 (4H, t, J=5, CH₂O); 5.62 (1H, s, H-4); 7.37 (1H, m,Ar—H); 7.61 (1H, m, Ar—H); 7.86 (1H, m, Ar—H). HRMS m/z (EI): 245.1052(M⁺, Calcd 245.1052 for C₁₄H₁₅NO₃), 230, 188, 160, 134, 114, 106, 86,77. Anal. Calcd for C₁₄H₁₅NO₃.0.2H₂O: C, 67.55; H, 6.03; N, 5.63. Found:C, 67.65; H, 6.06; N, 5.16.

8-Methoxy-2-morpholin-4-yl-chromen-4-one (Compound 303)

Yellow solid. mp 165° C. IR: (KBr)/(cm⁻¹): 3085, 2949, 2857, 1638, 1599,1571, 1411, 1245, 1116, 773. ¹H NMR: δ (d₆-DMSO): 3.51 (4H, t, J=4.5,CH₂N); 3.81 (4H, t, J=4.5, CH₂O); 3.91 (3H, s, MeO); 5.48 (1H, s, H-4);7.06 (1H, m, Ar—H); 7.22 (1H, m, Ar—H); 7.6 (1H, m, Ar—H). HRMS m/z(EI): 261.0991 (M⁺, Calcd 261.1001 for C₁₄H₁₅NO₄), 204, 151, 122, 114,107, 92.

7-Methoxy-2-(morpholin-4-yl)-chromen-4-one (Compound 304)

Off-white powder: mp 174-175° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.57 (4H,m); 3.81 (4H, m); 3.94 (3H, s); 5.50 (1H, s); 7.03 (1H, dd); 7.16 (1H,dd); 7.90 (1H, d). ¹³C NMR (50 MHz., d⁶-DMSO) δ 56.17; 65.66; 86.07;100.65; 113.36; 116.39; 126.15; 155.15; 162.63; 162.95; 175.39 ESMSm/z=261 (M+), 204

Compound 305 and 306

Compound Substituent Mw 305

380.16

Compound Substituent Mw 306

380.21

Synthesis of Starting Material Methyl 2,3-dihydroxybenzoate

Prepared from 2,3-Dihydroxybenzoic acid (1 g, 7.25 mmol), affording apale brown solid (0.29 g, 1.73 mmol, 23% yield); mp 81.1-81.9° C.;Rf=0.78 (solvent 95% DCM: 5% methanol); ¹H NMR (300 MHz, CDCl₃) δ 7.35(1H, d, Ar4), 7.15 (1H, d, Ar6), 6.85 (1H, dd, Ar5), 4.00 (3H, d, CH₃).

Preparation of 2-Hydroxy-3-trifluoromethanesulfonyloxy-benzoic acidmethyl ester

To a sample of methyl 2,3-dihydroxybenzoate (4.00 g, 23.80 mmol)dissolved in dichloromethane (25 ml), pyridine (0.96 ml, 11.9 mmol) wasadded and dimethylaminopyridine (0.07 g, 0.58 mmol). The mixture wascooled to 0° C. and trifluoromethane sulfonic anhydride (4.40 ml, 26.18mmol) was added dropwise by syringe. The reaction mixture was warmed toroom temperature and left to stir for 60 h. The organic layer washedwith 1M HCl (40 ml), dried (Na₂SO₄) and concentrated to dryness invacuo. The solid was recrystallized from ethyl acetate to yield whitecrystals. (2.62 g, 8.73 mmol, 37% yield), mp 91.8-92.3° C.; Rf=0.89(solvent; 95% DCM: 5% methanol); ES+(m/e) 300.00 (M+1); HPLC retentiontime=7.47 min (long); ¹H NMR (300 MHz, CDCl₃) δ7.85 (1H, d, Ar4), 7.45(1H, d, Ar6), 6.95 (1H, t, Ar5), 4.00 (3H, d, CH₃)

Preparation of 2-Hydroxy-4-trifluoromethanesulfonyloxy-benzoic acidmethyl ester

Prepared as for 2-Hydroxy-3-trifluoromethanesulfonyloxy-benzoic acidmethyl ester, from methyl 2,4-dihydroxybenzoate affording a whitecrystalline solid. ES+(m/e) 300.00 (M+1)

(b) Trifluoro-methanesulfonic acid2-hydroxy-3-(3-morpholin-4-yl-3-oxo-propionyl)-phenyl ester

Prepared from 2-Hydroxy-3-trifluoromethanesulfonyloxy-benzoic acidmethyl ester (2.10 g, 7 mmol), affording a pale brown solid (1.10 g,2.54 mmol, 36% yield). ES+(m/e) 398.25; ¹H NMR (300 MHz, CDCl₃) δ7.85(1H, d, Ar4), 7.35 (1H, d, Ar6), 6.90 (1H, dd, Ar5), 4.05 (2H, s, CH₂O),3.50 (8H, m, CH₂N, CH₂O).

(c) Trifluoro-methanesulfonic acid2-morpholin-4-yl-4-oxo-4H-chromen-8-yl ester (Compound 305)

Prepared from Trifluoromethanesulfonic acid2-hydroxy-3-(3-morpholin-4-yl-3-oxo-propionyl)-phenyl ester (0.91 g, 2.3mmol), affording a white solid (0.25 g, 0.662 mmol, 28.79% yield) mp177.8-178.9° C. Rf=0.30 (5% MeOH: 95% DCM). ES+(m/e) 380.16 (M+1). ¹HNMR (300 MHz, CDCl₃) δ3.50 (4H, m, CH₂N); 3.78 (4H, m, CH₂O); 5.46 (1H,s, Ar3); 7.40 (2H, m, Ar6, 7); 8.09 (1H, m, Ar5).

Trifluoro-methanesulfonic acid 2-morpholin-4-yl-4-oxo-4H-chromen-7-ylester (Compound 306)

Prepared from Trifluoromethanesulfonic acid3-hydroxy-4-(3-morpholin-4-yl-3-oxo-propionyl)-phenyl ester (1.50 g,3.80 mmol), affording a white solid (0.69 g, 1.83 mmol, 48% yield) mp143-145° C.; ES+(m/e)=380.21 (M+1); ¹H NMR (300 MHz, CDCl₃) δ3.45 (4H,m, CH₂N); 63.77 (4H, m, CH₂O); 65.36 (1H, s, CH); δ7.32 (2H, m); δ8.01(1H, m).

Further Derivatisation 7-Hydroxy-2-(morpholin-4-yl)-chromen-4-one(Compound 307)

To a mixture of7-(2,6-dichlorobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (6.60 g, 16.2mmol) (299) and 10% Pd/C (150 mg) was added methanol (150 ml), undernitrogen. The suspension was stirred under an atmosphere of hydrogen for40 h. The catalyst was removed by filtration through Celite, washingwith methanol. The solvent was removed by evaporation in vacuo toprovide an off-white solid. This was treated with fresh catalyst,re-suspended in methanol under nitrogen and stirred under an atmosphereof hydrogen for a further 72 h. The catalyst was removed by filtrationthrough Celite, washing with methanol. The filtrate was evaporated invacuo and the crude product re-crystallised from methanol to provide2.26 g (9.1 mmol, 57%) of the desired compound as a white solid. mp>250°C. (decomp). ¹H NMR (200 Mhz, d₆-DMSO) δ 3.78 (4H, m); 3.86 (4H, m);6.15 (1H, s); 7.05-7.13 (2H, m); 7.93 (1H, d); 11.3 (1H, bs). ESMSm/z=247 (M+), 190, 105.

Route 7a

Examples of compounds synthesised using synthetic route 7a are listed inthe following table. All examples of compounds synthesised by this routewere isolated with a purity of at least 99%.

Mw Compound Substituent LC-MS 308

310.24

Mw Compound Structure LC-MS 309

310

(a) 4-Hydroxy-benzo[f]-chromen-2-thione

Prepared from 2-hydroxy-1-acetonaphthone (3.72 g, 20.0 mmol) affording1.96 g (8.6 mmol, 13% yield) as a yellow solid. ¹H NMR (200 MHz.,d⁶-DMSO) δ 6.97 (1H, s); 7.73-7.90 (3H, m); 8.20 (1H, d); 8.40 (1H, d);9.43 (1H, d). EIMS m/z=228 (M+); 209; 170; 142; 69.

4-Hydroxy-benzo-[h]-chromen-2-thione

Prepared from 1-hydroxy-2-acetonaphthone (3.72 g, 20 mmol) affording1.09 g (5.32 mmol, 29% yield) as orange crystals. mp 221-223° C. ¹H NMR(200 MHz, d⁶-DMSO) δ 4.21 (1H, bs); 6.89 (1H, s); 7.91 (2H, m); 7.99(2H, m); 8.18 (1H, m); 8.56 (1H, m)

6-Bromo-4-hydroxy-chromene-2-thione

Prepared from 5-Bromo-2-hydroxyacetophenone (4.30 g, 20 mmol), affordinga yellow powder (1.85 g 7.20 mmol, 36%); ES+(m/e)=258 (M⁺+1)

(b) 2-(Ethylthio)-benzo[f]-chromen-4-one

Yellow crystalline solid: mp 126-127° C. IR (KBr) 1632; 1437; 815 cm⁻¹.¹H NMR (200 MHz., d⁶-DMSO) δ 1.48 (3H, t, CH₂CH₃); 3.32 (2H, q, CH₂CH₃);6.62 (1H, s, 3-H); 7.73-7.91 (3H, m); 8.19 (1H, d); 8.41 (1H, d); 10.01(1H, d). EIMS m/z=256 (M+); 170; 142. Anal. Calcd for C₁₅H₁₂O₂S.0.1H₂O:C, 69.80; H, 4.76. Found: C, 69.77; H, 4.53.

2-Ethylsulphanyl-benzo-[h]-chromen-4-one

Pale brown crystals (0.42 g, 2.85 mmol, 62% yield). mp 116-117° C. ¹HNMR (200 Mhz, d⁶-DMSO) δ1.45 (3H, t, J=7.4 Hz); 3.13 (2H, q, J=7.4 Hz);6.36 (1H, s); 7.65 (4H, m); 8.06 (1H, m); 8.41 (1H, m)

6-Bromo-2-ethylsulfanyl-chromen-4-one

Prepared from 6-Bromo-4-hydroxy-chromene-2-thione (0.57 g, 2.21 mmol),ethyl iodide (0.65 ml, 8 mmol) and potassium carbonate (0.35 g, 2.5mmol) affording a yellow solid (0.40 g, 1.40 mmol, 63%); ES+(m/e)=287(M⁺+1)

(c) 2-(Morpholin-4-yl)-benzo[f]-chromen-4-one (Compound 310)

Prepared from 2-(ethylthio)-benzo[f]-chromen-4-one (0.512 g, 2.0 mmol).Recrystallisation from methanol provided 0.238 g (0.84 mmol, 42% yield)of an off-white crystalline solid: mp 213-214° C. IR (KBr): 2956; 2861;1639; 1601; 1590; 1567; 1512; 1420; 1252; 1246; 1115; 821 cm⁻¹. ¹H NMR(200 MHz., d⁶-DMSO) δ 3.64 (4H, m); 3.86 (4H, m); 5.78 (1H, s,chromenone 3-H); 7.67-7.84 (3H, m); 8.14 (1H, d); 8.32 (1H, d); 10.16(1H, d). EIMS m/z=281 (M+); 253; 224; 196; 170.

6-Bromo-2-morpholin-4-yl-chromen-4-one (Compound 308)

Prepared from 6-Bromo-2-ethylsulfanyl-chromen-4-one (0.375 g, 1.35 mmol)and morpholine (0.54 ml, 6.25 mmol), affording a pale yellow solid.(0.0354 g, 1.14 mmol, 84%); m.p. 147-149° C.; ES+(m/e)=310.24 (M⁺+1);(200 MHz, CDCl₃) δ3.44 (4H, m); 3.77 (4H, m); 5.42 (1H, s); 7.11 (1H,d); 7.57 (1H, dd); 8.20 (1H, d)

2-(2,6-cis-dimethyl-morpholin-4-yl)-benzo[h]chromen-4-one (Compound 309)

Off white solid (0.174 g, 0.56 mmol, 56%): m.p. 211-212.5° C.; ES+(m/e)310 (M+1); R_(f)=0.30 (5% Methanol/DCM); ¹H NMR (200 MHz, CDCl₃) δ1.27(6H, d); 2.74 (2H, t); 3.72 (2H, m); 3.86 (2H, d); 5.56 (1H, s); 7.58(2H, m); 7.67 (1H, d); 7.86 (1H, m); 8.08 (1H, d); 8.19 (1H, m)

(d) 2-piperazin-1-yl-benzo[h]chromen-4-one (Compound 311)

Prepared from 2-ethylsulphanyl-benzo[h]chromen-4-one (0.384 g, 1.5 mmol)and piperazine (1.29 g, 15 mmol). Recrystallisation from ethyl acetateprovided an off white solid. (0.121 g, 0.43 mmol, 28% yield) mp 208-209°C. UV λ_(max)=317.0, 273.0, 255.0, 216.5 nm (Methanol). ¹H NMR (200 MHz,CDCl₃) δ 3.01 (4H, m); 3.55 (4H, m); 5.57 (1H, s); 7.56 (2H, m); 7.66(1H, d); 7.85 (1H, m); 8.08 (1H, d); 8.21 (1H, m). EIMS m/z (EI⁺): 280(M⁺), 261, 238, 225, 170, 139. Anal. Calcd for C₁₇H₁₆N₂O₂.0.3H₂O: C,71.46; H, 5.81; N, 9.80. Found: C, 71.88; H, 5.91; N, 9.33.

2-(Pyrrolidinyl)-benzo[h]chromen-4-one (Compound 312)

Off white solid. (0.104 g, 0.39 mmol, 26% yield) mp 234-236° C. ¹H NMR(200 MHz, CDCl₃) δ 2.05 (4H, m); 3.55 (4H, m); 5.36 (1H, s); 7.55 (2H,m); 7.65 (1H, d); 7.83 (1H, m); 8.10 (1H, d); 8.19 (1H, m). EIMS m/z(EI⁺): 265 (M⁺), 210, 196, 170, 114, 95. Anal. Calcd forC₁₇H₁₅NO₂.0.28CH₂Cl₂: C, 71.70; H, 5.42; N, 4.84. Found: C, 71.43; H,5.76; N, 4.75.

2-(3-Hydroxymethyl-piperidin-1-yl)-benzo[h]chromen-4-one (Compound 313)

Off white solid. (0.131 g, 0.42 mmol, 43% yield) mp 209-210° C. UVλ_(max)=319.0, 284.0, 274.0, 254.0, 217.0 nm (Methanol). FT-IR 3300,2924, 2854, 1640, 1609, 1559, 1439 cm⁻1. ¹H NMR (200 MHz, CDCl₃) δ 1.30(1H, m); 1.79 (4H, m); 3.14 (2H, m); 3.51 (1H, m); 3.65 (1H, m); 3.98(1H, m); 4.14 (1H, m); 5.64 (1H, s); 7.49 (2H, m); 7.60 (1H, d); 7.77(1H, m); 8.02 (1H, d); 8.17 (1H, m). EIMS m/z (EI⁺): 309 (M⁺), 292, 278,224, 196, 170, 138, 82, 55. Anal. Calcd for C₁₉H₁₉NO₃.0.1H₂O: C, 73.34;H, 6.32; N, 4.50. Found: C, 73.28; H, 6.19; N, 4.13

2-(4-Methyl-piperazin-1-yl)-benzo[h]chromen-4-one (Compound 314)

White solid. (0.194 g, 0.66 mmol, 67% yield) mp 184-185° C. UVλ_(max)=316.0, 272.0, 254.5, 218.0 nm (Methanol). ¹H NMR (200 MHz,CDCl₃) δ 2.32 (3H, s); 2.54 (4H, t); 3.60 (4H, t); 5.59 (1H, s); 7.56(2H, m); 7.67 (1H, d); 7.83 (1H, m); 8.08 (1H, d); 8.21 (1H, m). EIMSm/z (EI⁺): 294 (M⁺), 237, 224, 210, 196, 170, 139, 123, 70. Anal. Calcdfor C₁₈H₁₈N₂O₂.1H₂O. 0.1CH₃OH: C, 68.85; H, 6.52; N, 8.88. Found: C,68.63; H, 6.45; N, 8.57.

2-(3-Hydroxy-pyrollidin-1-yl)-benzo[h]chromen-4-one (Compound 315)

White solid. (0.201 g, 0.72 mmol, 72% yield) mp 256-257° C. UVλ_(max)=318, 283.5, 273.0, 253.0, 215.0 nm (Methanol). ¹H NMR (200 MHz,DMSO) 62.18 (2H, m); 3.45 (4H, m); 4.58 (1H, m); 5.32 (1H, m) 5.41 (1H,s); 7.83 (2H, m); 7.93 (1H, d); 8.05 (1H, d); 8.16 (1H, m); 8.45 (1H,m). EIMS m/z (EI⁺): 281 (M⁺), 264, 236, 224, 210, 196, 181, 170, 139,114, 67. Anal. Calcd for C₁₇H₁₅NO₃.0.2H₂O: C, 71.67; H, 5.45; N, 4.92Found: C, 71.65; H, 5.34; N, 4.49

2-[(Tetrahydrofuran-2-ylmethyl)-amino]-benzo[h]chromen-4-one (Compound316)

Off white crystalline solid. (0.107 g, 0.36 mmol, 37% yield) mp 139-140°C. UV %=314.0, 280.5, 270.5, 252.5, 216.5 nm (Methanol). ¹H NMR (200MHz, CDCl₃) δ 1.65 (1H, m); 1.91 (3H, m); 3.14 (2H, m); 3.21 (1H, m);3.38 (1H, M); 3.81 (2H, m); 4.11 (1H, m); 5.40 (1H, t); 5.47 (1H, s);7.54 (2H, m); 7.65 (1H, d); 7.82 (1H, m); 8.08 (1H, d); 8.24 (1H, m).EIMS m/z (EI⁺): 295 (M⁺), 272, 225, 211, 196, 186, 171, 158, 84, 71.Anal. Calcd for C₁₆H₁₇NO₃.0.3H₂O: C, 71.85; H, 5.90; N, 4.66. Found: C,72.12; H, 5.80; N, 4.33

2-(2-Methyl-morpholin-4-yl)-benzo[h]chromen-4-one (Compound 317)Synthesis of 2-Methyl morpholine

Ref: Bettoni et al. Tetrahedron, 1980, 36, 409-415

(i) 1-(2-Hydroxy-ethylamino)-propan-2-ol

Propylene oxide (2.32 g, 0.04 mmol) was added dropwise to a solution ofethanolamine (10.0 g, 0.16 mmol) in water (50 ml) at 0° C., and thesolution stirred at room temperature for 5 h. Water was removed byevaporation in vaccuo resulting in a colourless oil which was thendistilled under reduced pressure to yield the title compound as acolourless oil. (3.61 g, 30.34 mmol, 76%) ¹H NMR (200 MHz, CDCl₃) δ1.15(3H, d); 2.46 (2H, m); 2.71 (2H, t,); 3.62 (2H, t); 3.90 (1H, m,); 4.10(3H, s.).

(ii) Toluene-4-sulfonic acid2-[(2-hydroxy-propyl)-(toluene-4-sulfonyl)-amino]-ethyl ester

Tosyl chloride (11.60 g, 60.80 mmol) was added in small portions to astirred solution of 1-(2-Hydroxy-ethylamino)-propan-2-ol (3.60 g, 30.25mmol) in anhydrous pyridine at 0° C. The reaction was stirred at roomtemperature for 24 h and then poured onto ice-water (200 ml). Themixture was extracted into DCM (100 ml). The organic extract washed with2N HCl, water, and was evaporated in vaccuo to give a brown residuewhich was used without further purification.

(iii) 2-Methyl-4-(toluene-4-sulfonyl)-morpholine

Sodium hydroxide (0.91 g, 0.02 mol) suspended in methanol (15 ml) wasadded to a stirred solution of Toluene-4-sulfonic acid2-[(2-hydroxy-propyl)-(toluene-4-sulfonyl)-amino]-ethyl ester (9.69 g,0.02 mol) in DCM (15 ml). After 1 h, water (50 ml) was added to thesolution. The organic layer was collected, dried over sodium sulphateand evaporated in vaccuo to yield a green oily residue. This waspurified by chromatographic separation (20% Ethyl acetate:petrol) toyield the title compound as a white solid. (1.70 g, 6.66 mmol, 33%).

(iv) 2-Methyl Morpholine

2-Methyl-4-(toluene-4-sulfonyl)-morpholine (1.65 g, 6.51 mmol) wasdissolved in warm pentanol (30 ml). The solution was cooled to roomtemperature and sodium (1.49 g, 65 mmol) was added in small portions.The reaction mixture was stirred vigorously and heated to reflux for 3h. Upon cooling, water (50 ml) was added. The two layers were separated,the aqueous layer was extracted with ether, and this in turn wasextracted with 2N HCl. The alcoholic solution was extracted with 2N HCl.The combined acidic solutions were then made alkaline by addition ofsodium hydrogen carbonate, and continuously extracted with ether. Theether was evaporated in vaccuo to yield the title compound as acolourless oil. (0.517 g, 5.11 mmol, 79%) ¹H NMR (200 MHz, CDCl₃) δ1.15(3H, d); 2.74 (5H, m); 3.81 (4H, m).

Final Compound (Compound 317)

Off white crystalline solid. (0.085 g, 0.29 mmol, 20% yield). mp181-183° C. UV λ=214.4, 217.4 (λ_(max)), 255.0, 272.8, 281.8, 300.8,315.2 nm (Methanol). FT-IR (cm⁻¹)=3174, 2976, 2860, 1614, 1557, 1388,1245, 1086, 795, 747. ¹H NMR (200 MHz, CDCl₃) δ1.25 (3H, d); 2.81 (1H,t); 3.16 (1H, dt); 3.71 (2H, m); 3.83 (2H, t); 4.02 (1H, m); 5.55 (1H,s); 7.55 (2H, m); 7.66 (1H, d); 7.83 (1H, d); 8.06 (1H, d); 8.17 (1H,d). ESMS m/e=296 (M+1). Anal. Calcd. For C₁₈H₁₇NO₃. 0.1H₂O: C, 72.76; H,5.83; N, 4.71. Found: C, 72.74; H, 5.77; N, 4.60.

(f) 2-(4-Hydroxymethyl-piperidin-1-yl)-benzo[h]chromen-4-one (Compound318)

Prepared from(Benzo-[h]-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneresin and 4-piperidine methanol (0.0027 g, 0.036 mmol). Productobtained=0.0039 g. m/z (ES⁺): 310 (M+1) 5% Methanol/DCM, R_(f)=0.21

2-[(2-Hydroxy-2-phenyl-ethyl)-methyl-amino]benzo[h]chromen-4-one(Compound 319)

m/z (ES⁺): 346 (M+1); 5% Methanol/DCM, R_(f)=0.30

2-(3-Diethylamino-propylamino)-benzo[h]chromen-4-one (Compound 320)

m/z (ES⁺): 325 (M+1); 5% Methanol/DCM, R_(f)=0.19

2-((S)-2-Hydroxymethyl-pyrrolidin-1-yl)-benzo[h]chromen-4-one (Compound321)

m/z (ES⁺): 296 (M+1); 5% Methanol/DCM, R_(f)=0.29

2-(3-Methoxy-propylamino)-benzo[h]chromen-4-one (Compound 322)

m/z (ES⁺): 284 (M+1); 5% Methanol/DCM, R_(f)=0.32

2-(1-Benzyl-piperidin-4-ylamino)-benzo[h]chromen-4-one (Compound 323)

m/z (ES⁺): 385 (M+1); 5% Methanol/DCM, R_(f)=0.17

2-(Cyclopentylamino)-benzo[h]chromen-4-one (Compound 324)

m/z (ES⁺): 280 (M+1); 5% Methanol/DCM, R_(f)=0.33

2-(2,2-Dimethoxy-ethylamino) benzo[h]chromen-4-one (Compound 325)

m/z (ES⁺): 300 (M+1); 5% Methanol/DCM, R_(f)=0.29

2-Butylamino-benzo[h]chromen-4-one (Compound 326)

m/z (ES⁺): 268 (M+1); 5% Methanol/DCM, R_(f)=0.30

2-(2-Trifluoromethyl-benzylamino)-benzo[h]chromen-4-one (Compound 327)

m/z (ES⁺): 370 (M+1); 5% Methanol/DCM, R_(f)=0.31

2-(3-Hydroxy-propylamino)-benzo[h]chromen-4-one (Compound 328)

m/z (ES⁺): 270 (M+1); 5% Methanol/DCM, R_(f)=0.12

2-(2-Hydroxy-2-phenyl-ethylamino)-benzo[h]chromen-4-one (Compound 329)

m/z (ES⁺): 332 (M+1); 5% Methanol/DCM, R_(f)=0.22

2-(Thiazolidin-3-yl)-benzo[h]chromen-4-one (Compound 330)

m/z (ES⁺): 284 (M+1); 5% Methanol/DCM, R_(f)=0.35

2-(2-Hydroxy-propylamino)-benzo[h]chromen-4-one (Compound 331)

m/z (ES⁺): 270 (M+1); 5% Methanol/DCM, R_(f)=0.15

2-[(2-Hydroxy-ethyl)methyl-amino]-benzo[h]chromen-4-one (Compound 332)

m/z (ES⁺): 270 (M+1); 5% Methanol/DCM, R_(f)=0.19

2-(Ethyl-hyroxymethyl-amino)benzo[h]chromen-4-one (Compound 333)

m/z (ES⁺): 284 (M+1); 5% Methanol/DCM, R_(f)=0.25

2-(Dibutylamino)-benzo[h]chromen-4-one (Compound 334)

m/z (ES⁺): 324 (M+1); 5% Methanol/DCM, R_(f)=0.26

2-(2-Methoxy-ethylamino)-benzo[h]chromen-4-one (Compound 335)

m/z (ES⁺): 270 (M+1); 5% Methanol/DCM, R_(f)=0.10

2-(Isopropylamino)-benzo[h]chromen-4-one (Compound 336)

m/z (ES⁺): 254 (M+1); 5% Methanol/DCM, R_(f)=0.27

Route 7b 2-Hydroxy-4-(4-methoxybenzyloxy)-acetophenone

A mixture of 2,4-dihydroxyacetophenone (7.30 g, 48 mmol), potassiumcarbonate (7.30 g, 53 mmol) and sodium iodide (0.75 g, 5.0 mmol) inanhydrous acetonitrile (60 ml) was treated with 4-methoxybenzyl chloride(6.5 ml, 48 mmol). The mixture was heated to 65° C. and stirred for 16h. The mixture was treated with 1M hydrochloric acid (120 ml) andextracted into ethyl acetate (120 ml). The ethyl acetate extract washedwith 1M hydrochloric acid (100 ml) and brine (100 ml), dried over sodiumsulphate and evaporated in vacuo. The crude product was stirredvigorously in ether and filtered to provide 6.31 g (23.4 mmol, 49%yield) of the title compound as a beige powder.

(a) 4-Hydroxy-7-(4-methoxybenzyloxy)-chromen-2-thione

Prepared from 2-hydroxy-4-(4-methoxybenzyloxy)-acetophenone (5.44 g, 20mmol) affording 2.04 g (6.5 mmol, 32% yield) as a yellow powder.

(e)S-(7-(Hydroxy)-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneresin

Prepared from Merrifield resin (1% cross-linked, 1.2 mmol/g) (0.70 g,0.84 mmol) and a solution of4-hydroxy-7-(4-methoxybenzyloxy)-chromen-2-thione (0.70 g, 2.2 mmol) inDMF (3 ml).

(g, followed by f(i)(ii)) 7-(Benzyloxy)-2-(morpholin-4-yl)-chromen-4-one(Compound 337)

Prepared fromS-(7-(Hydroxy)-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneresin (0.030 g) affording 0.0014 g (0.004 mmol) as a crude residue.

7-(4-Cyanobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 338)

Estimated 88% pure by LC-MS; ESMS m/z=363 (M+1)⁺.

Methyl 4-(2-(morpholin-4-yl)-4-oxo-4H-chromen-7-yloxymethyl)-benzoate(Compound 339)

Estimated 74% pure by LC-MS; ESMS m/z=396 (M+1)⁺.

Methyl 3-(2-(morpholin-4-yl)-4-oxo-4H-chromen-7-yloxymethyl)-benzoate(Compound 340)

Estimated 82% pure by LC-MS; ESMS m/z=396 (M+1)⁺.

7-(3-Chlorobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 341)

Estimated 90% pure by LC-MS; ESMS m/z=374, 372 (M+1)⁺.

7-(3-Methylbenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 342)

Estimated 86% pure by LC-MS; ESMS m/z=352 (M+1)⁺.

Examples of compounds synthesised using a variant of route 7b in which a2,5-dihydroxyacetophenone starting material was used in place of2,4-dihydroxyacetophenone include the following:

6-Hydroxy-2-(morpholin-4-yl)-chromen-4-one (Compound 343)

S-(6-(Hydroxy)-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneresin (0.030 g, <0.036 mmol) was swelled in DCM (2 ml). After shakingfor 10 minutes the mixture was treated with m-chloroperbenzoic acid (0.2g, 1.1 mmol). The mixture was shaken at room temperature for 3 h andthen filtered. The resin washed in order with DCM, methanol, DCM andre-suspended in DCM (2 ml). After shaking for 15 minutes the mixture wastreated with a solution of morpholine (0.005 ml, 0.05 mmol) in DCM (2ml). The mixture was shaken at room temperature for 16 h and filtered,washing the resin with DCM and methanol. The filtrate was evaporated invacuo to provide the crude title compound. The product was submitted foranalysis for LC-MS without further purification. Estimated >95% pure byLC-MS; ESMS m/z=248 (M+1)⁺.

((g) followed by (f) (i) (ii))6-(4-Cyanobenzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 344)

Prepared fromS-(6-(Hydroxy)-4-oxo-4H-chromen-2-yl)-thiomethylpolystyrene-divinylbenzeneresin (0.030 g) affording a crude residue. Estimated 80% pure by LC-MS;ESMS m/z=363 (M+1)⁺.

N-[3-(2-(morpholin-4-yl)-4-oxo-4H-chromen-6-yloxy)-propyl]-phthalimide(Compound 345)

Estimated 66% pure by LC-MS; ESMS m/z=435 (M+1)⁺.

Route 7b(i)

Examples of compounds formed using synthetic route 7b(i) are listed inthe following tables.

Mw Compound R R Substituent LC-MS 346 Phenyl 4-Br 432 347 Phenyl 4-t-Bu394 348 Phenyl 4-OMe 382 349 Phenyl — 352 350 Pyridin-4-yl N—O⁻ 369 351Pyridin-2-yl N—O⁻ 355

Mw Compound R R Substituent LC-MS 352 Phenyl 2-Cl 404 353 Phenyl 4-Cl404 354 Napth-2-yl — 418 355 Phenyl — 368 356 Ethyl — 320

Mw Compound R R Substituent LC-MS 357 Phenyl 3-OMe 368 358 Phenyl3-N(═O)O⁻ 383 359 Phenyl 3-F 356 360 Phenyl 3,4-di-F 374 361 Phenyl 4-Me352 362 Phenyl 4-t-Bu 394 363 Phenyl 3-Br 417 364 Pyridin-3-yl N—O⁻ 355365 Pyridin-4-yl N—O⁻ 355

Mw Compound R R Substituent LC-MS 366 Phenyl — 416 367 Ethyl — 368 368Methyl Phenyl 430

Route 7c

Examples of compounds formed using synthetic route 7c are listed in thetables below.

Substituent Mw Compound position Substituent LC-MS 369 2

414 370 2

384 371 4

392 372 3

384 373 2

338 374 2

365 375 3

467

Mw Compound Substituent LC-MS 376

364 377

314 378

352 379

446

Substituent Mw Compound position Substituent LC-MS 380 4

442 381 3

414 382 4

414 383 4 *—C≡N 333 384 4

350 385 4

338 386 2

384 387 4

392 388 3

392 389 3

384 390 3

380 391 4 *—Cl 342 392 2

338 393 3

338 394 4

338 395 3 *—OH 324 396 4 *—OH 324 397 4

365 398 2

376 399 3

467

Mw Compound Substituent LC-MS 400

372 401

334 402

364 403

314 404

314 405

358 406

298 407

352 408

364 409

356 410

398 411

347 412

430 413

414

Route 8 7-(Benzyloxy)-2-(morpholin-4-yl)-chromen-4-one (Compound 337)

Prepared from benzyl bromide (0.25 ml, 2.0 mmol). Recrystallisation frommethanol provided 0.098 g (0.29 mmol, 58% yield) as white crystals: mp170-172° C. UV λ_(max)=258.0, 310.5 nm (methanol). ¹H NMR (200 MHz.,d⁶-DMSO) δ 3.59 (4H, m); 3.82 (4H, m); 5.31 (2H, s, CH₂); 5.52 (1H, s,3-H); 7.13 (1H, dd, J=2.3, 8.7 Hz., 6-H); 7.28 (1H, d, J=2.3 Hz., 8-H);7.45-7.60 (5H, m); 7.91 (1H, d, J=8.7 Hz., 5-H). ESMS m/z=338 (M+), 179.Anal. Calcd for C₂₀H₁₉NO₄: C, 71.20; H, 5.68; N, 4.15. Found: C, 71.15;H, 5.63; N, 3.85.

7-(4-Fluorobenzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound 414)

White crystals: mp 201-203° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.60 (4H,m); 3.82 (4H, m); 5.29 (2H, s, CH₂); 5.52 (1H, s, 3-H); 7.13 (1H, m,6-H); 7.29 (1H, m); 7.34 (2H, m); 7.64 (2H, m, 8-H); 7.92 (1H, m, 5-H).ESMS m/z=344 (M+)

7-(4-Chlorobenzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound 415)

White crystals: decomp. >185° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.60 (4H,m); 3.82 (4H, m); 5.31 (2H, s, CH₂); 5.52 (1H, s, 3-H); 7.13 (1H, dd,J=2.2, 8.7 Hz., 6-H); 7.28 (1H, d, J=2.2 Hz., 8-H); 7.59-7.71 (4H, m);7.92 (1H, d, J=8.7 Hz., 5-H). ESMS m/z=371, 373 (M+)

7-(4-Bromobenzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound 416)

White crystals: mp 221-222° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.60 (4H,m); 3.82 (4H, m); 5.30 (2H, s, CH₂); 5.52 (1H, s, 3-H); 7.13 (1H, dd,J=2.0, 8.7 Hz., 6-H); 7.27 (1H, d, J=2.0 Hz., 8-H); 7.53 (2H, d, J=8.3Hz.); 7.72 (2H, d, J=8.3 Hz.); 7.92 (1H, d, J=8.7 Hz., 5-H). ESMSm/z=419, 417 (M+)

7-(2-Chlorobenzyloxy)-2-morpholin-4-yl-chromen-4-one (Compound 417)

White crystals: mp 167-168° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.61 (4H,m); 3.81 (4H, m); 5.36 (2H, s, CH₂); 5.54 (1H, s, 3-H); 7.15 (1H, dd,J=2.3, 8.7 Hz., 6-H); 7.35 (1H, d, J=2.3 Hz., 8-H); 7.50-7.76 (4H, m);7.93 (1H, d, J=8.7 Hz., 5-H). ESMS m/z=373, 371 (M+)

7-(Naphthalen-2-ylmethoxy)-2-morpholin-4-yl-chromen-4-one (Compound 418)

White crystals: mp 263-264° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 3.60 (4H,m); 3.81 (4H, m); 5.49 (2H, s, CH₂); 5.53 (1H, s, 3-H); 7.19 (1H, dd,J=2.2, 8.7 Hz., 6-H); 7.34 (1H, d, J=2.2 Hz., 8-H); 7.62-7.73 (3H, m);7.92 (1H, d, J=8.7 Hz., 5-H); 8.02-8.11 (4H, m). ESMS m/z=387 (M+)

7-Cyclohexylmethoxy-2-(morpholin-4-yl)-chromen-4-one (Compound 419)

White crystals: mp 187-188° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 1.16 (5H, m,cyclohexyl); 1.87 (6H, m, cyclohexyl); 3.60 (4H, m, morpholine); 3.80(4H, m, morpholine); 3.97 (2H, s, CH₂); 5.50 (1H, s, 3-H); 7.12 (1H, dd,J=2.1, 8.7 Hz., 6-H); 7.18 (1H, d, J=2.1 Hz., 8-H); 7.88 (1H, d, J=8.7Hz., 5-H). MS (ES+) m/z=344 (M+)

7-Propoxy-2-(morpholin-4-yl)-chromen-4-one (Compound 420)

White crystals: decomp. >115° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 1.08 (3H,t, CH₂CH₂CH₃); 1.86 (2H, m, CH₂CH₂CH₃); 3.60 (4H, m); 3.81 (4H, m); 4.12(2H, t, CH₂CH₂CH₃); 5.51 (1H, s, 3-H); 7.04 (1H, dd, J=2.0, 8.7 Hz.,6-H); 7.18 (1H, d, J=2.0 Hz., 8-H); 7.89 (1H, d, J=8.7 Hz., 5-H). ESMSm/z=290 (M+)

N-[2-(2-(Morpholin-4-yl)-4-oxo-4H-chromen-7-yloxy)-ethyl]-phthalimide(Compound 421)

White crystals: decomp. >230° C. ESMS m/z=421 (M+)

N-[3-(2-(Morpholin-4-yl)-4-oxo-4H-chromen-7-yloxy)-propyl]-phthalimide(Compound 422)

White crystals: mp 210-211° C. ¹H NMR (200 MHz., d⁶-DMSO) δ 2.60 (2H, m,NCH₂CH₂CH₂O); 3.58 (4H, m, morpholine); 3.81 (4H, m, morpholine); 3.89(2H, m, NCH₂CH₂CH₂O); 4.22 (2H, m, NCH₂CH₂CH₂O); 5.50 (1H, s, 3-H); 6.86(1H, dd, J=2.0, 8.6 Hz., 6-H); 7.03 (1H, d, J=2.0 Hz., 8-H); 7.83 (1H,d, J=8.6 Hz., 5-H); 7.95 (4H, m, phth-H₄). ESMS m/z=435 (M+).

Route 9 7-Benzoyloxy-2-(morpholin-4-yl)-chromen-4-one (Compound 423)

Prepared from benzoyl chloride (0.13 ml, 1.1 ml). Recrystallisation fromethyl acetate provided 0.19 g (0.55 mmol, 55%) as white crystals: mp204-206° C. Anal. Calcd for C₂₀H₁₇NO₅: C, 68.37; H, 4.88; N, 3.99.Found: C, 68.14; H, 4.87; N, 3.73. UV_(max)=258.0, 311.0 nm (methanol).¹H NMR (200 MHz., d⁶-DMSO) δ 3.64 (4H, m); 3.83 (4H, m); 5.65 (1H, s,3-H); 7.45 (1H, m); 7.74 (3H, m); 8.87 (1H, m); 8.09 (1H, m); 8.26 (2H,m). MS (ES) m/z=352 (M⁺); 179.

Further Synthesis Details2-(2,3-dihydro-benzo[1,4]oxazin-4-yl)-benzo[h]chromen-4-one (Compound424)

Synthesis of 3,4-dihydro-2H-benzo[1,4]oxazine

a) N-(tertbutoxycarbonyl)-2-aminophenol

A mixture of 2-aminophenol (0.545 g, 5 mmol) anddi-tert-butyldicarbonate (1.86 g, 10 mmol) in anhydrous THF (20 ml) wasstirred at room temperature for 12 h. After concentration andhydrolysis, the aqueous layer was extracted with EtOAc (3×30 mL). Theorganic layer were combined and dried over MgSO₄ and the solvent wasremoved under reduce pressure. The crude product was purified bycrystallisation (petrol/ether 8/2).

The pure compound is obtained as a white solid (0.839 g, 86% yield).m.p=145° C.; R_(f)=0.28 (petrol/ether 8/2); LCMS m/z 196 ([M+1]⁺); ¹HNMR (200 MHz, CDCl₃): δ 1.61 (9H, s); 6.65 (1H, bs); 6.48-7.08 (4H, m);8.16 (1H, bs); ¹³C NMR (75 MHz, CDCl₃): (29.9 (3C); 83.7; 120.3; 122.5;122.9; 127.1; 127.3; 148.9; 156.7. IR (film): 3280; 1688; 1146 cm⁻¹.

b) N-(tertbutoxycarbonyl)-2,3-dihydro-benzo[1,4]oxazine

A solution of dry acetone (100 mL) containingN-(tertbutoxycarbonyl)-2-aminophenol (0.722 g, 3.69 mmol), potassiumcarbonate (10.2 g, 73.8 mmol) and 1,2-dibromobutane (2.54 mL, 29.6 mmol)was refluxed for 18 h. The reaction was monitored by TLC (petrol/ether8/2). After cooling, the mixture was filtered through celite. Afterconcentration and hydrolysis, the aqueous layer was extracted with EtOAc(3×40 mL), dried over MgSO₄ and the solvent was removed under reducepressure. The crude product was purified by flash chromatography onsilica gel (petrol/EtOAc 95/5) to yield the title compound as a whitesolid (0.70 g, 82%). m.p=78-79° C.; R_(f)=0.44 (petrol/ether 8/2); LCMSm/z 236 ([M+1]⁺); ¹H NMR (300 MHz, CDCl₃): δ 1.59 (9H, s); 3.87 (4H, m);4.26 (4H, m); 6.86-7.02 (4H, m); ¹³C NMR (75 MHz, CDCl₃): δ 27.4 (3C);41.1; 64.6 (2C); 80.6 (2C); 116.0; 119.2; 122.6; 123.4; 125.2; 144.9;151.6. IR (film): 2975; 1696; 1494; 1143 cm⁻¹.

Ref: Kubick et al. Eur. J. Org. Chem. 2001, 311-312

c) 3,4-dihydro-2H-benzo[1,4]oxazine

To a solution of dichloromethane (10 mL) containingN-(tertbutoxycarbonyl)-2,3-dihydro-benzo[1,4]oxazine (0.438, 1.86 mmol)at 0° C. was added slowly trifluoroacetic acid (1.0 mL, 7.44 mmol). Thereaction mixture was stirred at this temperature during 5 h, then thesolvent was removed in vaccuo. The crude product was dissolved in EtOAc(15 mL) and washed successively with 10% Na₂CO₃ solution and water. Theorganic layer was dried over MgSO₄ and the solvent was removed underreduce pressure. The title compound was obtained pure as brown oil(0.245 g, 98%). R_(f)=0.31 (petrol/ether 5/5); LCMS m/z 136 ([M+1]⁺); ¹HNMR (300 MHz, CDCl₃): δ 3.43 (4H, m); 3.54 (1H, s); 4.27 (4H, m);6.60-6.82 (4H, m); ¹³C NMR (75 MHz, CDCl₃) δ 43.4; 67.7; 118.1; 119.2;121.3; 123.7; 136.1; 146.6. IR (film): 3375; 1498; 741 cm⁻¹.

2-(2,3-dihydro-benzo[1,4]oxazin-4-yl)-benzo[h]chromen-4-one

To a solution of anhydrous THF (5 mL) containing3,4-dihydro-2H-benzo[1,4]oxazine (0.324 g, 1.6 mmol), at 0° C., wasadded dropwise n-BuLi (1.24 mL, 3.12 mmol, 2.5 N) while the temperatureof 0-10° C. was maintained. After stirring for 30 min at 0° C., thesulfone (0.436 g, 1.6 mmol) was added in THF solution (10 mL). Thereaction mixture was warmed slowly at rt and stirred for 20 h (TLCether). The mixture was poured into 10 mL of 2N HCl (10 mL) andextracted with dichloromethane (3×20 mL). The organic layers werecombined, dried over MgSO₄ and concentrated under reduce pressure. Thecrude product was purified by preparative HPLC to yield the titlecompound as a yellow solid (2 mg).

LCMS m/z 330 ([M+1]⁺); ¹H NMR (300 MHz, CDCl₃): δ 4.01 (2H, m, CH₂N),4.35 (2H, m, CH₂O), 6.06 (1H, s), 6.85-7.05 (4H, m, ArH), 7.44-8.28 (6H,m, ArH).

Ref: Wynberg et al. J. Org. Chem. 1993, 58, 5101-5106

Biological Examples DNA-PK Inhibition

In order to assess the inhibitory action of the compounds against DNA-PKin vitro, the following assay was used to determine IC₅₀ values.

Mammalian DNA-PK, isolated from Hela cell nuclear extract (Gell, D. andJackson S. P., Nucleic Acids Res. 27:3494-3502 (1999)), was incubatedwith Z buffer (25 mM Hepes (Sigma); 12.5 mM MgCl₂ (Sigma); 50 mM KCl(Sigma); 1 mM DTT (Sigma); 10% Glycerol (Sigma); 0.1% NP-40 (Sigma); pH7.4) in polypropylene 96 well plates and varying concentrations ofinhibitor added. All compounds were diluted in DMSO to give a finalassay concentration of between 10 and 0.001 μM, with DMSO being at afinal concentration of 1% per well. The total assay volume per well was40 μl.

After 10 minutes of incubation at 30° C. the reactions were initiated bythe addition of Na-ATP (50 μM final), ³³p-γATP and a 30mer doublestranded DNA oligonucleotide (10 ng/μl) in a volume of 10 μl. Designatedpositive and negative reaction wells were done in combination withcompound wells (unknowns) in order to calculate % enzyme activities. Theplates were then shaken for 2 minutes and incubated at 30° C. for 45minutes.

Following the incubation, the reactions were quenched by the addition of50 μl 30% acetic acid to each well. The plates were then shaken for 5minutes and the contents of each plate (80 μl from each well)transferred over to a 96 well Polyfiltronics filtration plate,containing P81-phosphocellulose membrane (TRADE MARK)(Whatman, UK). Thesolutions were vacuum pumped through the membrane and each well membranewashed four times using 300 μl of 15% acetic acid. The well membraneswere then air dried and 20 μl of scintillant was added to each well.

The plates were transferred to a TopCount NXT (TRADE MARK) (Packard, UK)for scintillation counting. Values recorded are counts per minute (cpm)following a 1 minute counting time for each well.

The enzyme activity for each compound is then calculated using thefollowing equation:

${\%\mspace{14mu}{Inhibition}} = {100 - \left( \frac{\left( {{{cpm}\mspace{14mu}{of}\mspace{14mu}{unknown}} - {{mean}\mspace{14mu}{negative}\mspace{14mu}{cpm}}} \right)\; \times 100}{\left( {{{mean}\mspace{14mu}{positive}\mspace{14mu}{cpm}} - {{mean}\mspace{14mu}{negative}\mspace{14mu}{cpm}}} \right)} \right)}$

The results are detailed below in Table 1 as IC₅₀ values (theconcentration at which 50% of the enzyme activity is inhibited). Theseare determined over a range of different concentrations, normally from10 μM down to 0.01 μM. Such IC₅₀ values are used as comparative valuesto identify increased compound potencies. LY294002 exhibited an IC₅₀ of1.5 μM.

Enhancement Ratio

The Enhancement Ratio (ER) is a ratio of the enhancement of cell growthinhibition elicited by the DNA-PK inhibitor after 2 Grays of irradiationcompared to untreated control cells. DNA-PK inhibitors were used at afixed concentration of 25 micromolar. Radiation was delivered by aFaxitron 43855D X-ray system at a dose rate of 1Gy per minute TheEnhancement ratio at 2 Gy irradiation was calculated from the formula:

${ER} = \frac{\begin{matrix}{{Cell}\mspace{14mu}{growth}\mspace{14mu}{in}\mspace{14mu}{presence}\mspace{14mu}{of}\mspace{14mu}{DNA}\text{-}{PK}\mspace{14mu}{inhibitor}\; \times} \\{{Cell}\mspace{14mu}{growth}\mspace{14mu}{after}\mspace{14mu}{IR}}\end{matrix}}{\begin{matrix}{{Cell}\mspace{14mu}{growth}\mspace{14mu}{of}\mspace{14mu}{untreated}\mspace{14mu}{cells} \times} \\{{Cell}\mspace{14mu}{growth}\mspace{14mu}{after}\mspace{14mu}{IR}\mspace{14mu}{in}\mspace{14mu}{presence}\mspace{14mu}{of}\mspace{14mu}{DNA}\text{-}{PK}\mspace{14mu}{inhibitor}}\end{matrix}}$

Cell growth was assessed using the sulforhodamine B (SRB) assay (Skehan,P., Storung, R., Scudiero, R., Monks, A., McMahon, J., Vistica, D.,Warren, J. T., Bokesch, H., Kenny, S. and Boyd, M. R. (1990) Newcalorimetric cytotoxicity assay for anticancer-drug screening. J. Natl.Cancer Inst. 82:1107-1112). 400 HeLa cells were seeded into each well ofa flat-bottomed 48-well microtiter plate in a volume of 200 μl andincubated for 6 h at 37° C. Cells were either replaced with media aloneor with media containing DNA-PK inhibitor at a final concentration of 25μM. Cells were allowed to grow for a further 1 h before irradiation ormock irradiation. Cells untreated with DNA-PK inhibitor or unirradiatedwere used as a control. Cells treated with DNA-PK inhibitor alone wereused to assess the growth inhibition by the DNA-PK inhibitor.

Cells were left for a further 16 h before replacing the media andallowing the cells to grow for a further 6 days at 37° C. The media wasthen removed and the cells fixed with 200 μl of ice cold 10% (w/v)trichloroacetic acid. The plates were incubated at 4° C. for 20 minutesand then washed four times with water. Each well of cells was thenstained with 200 μl of 0.4% (w/v) SRB in 1% acetic acid for 20 minutesbefore washing four times with 1% acetic acid. Plates were then driedfor 2 h at room temperature. The dye from the stained cells wassolubilized by the addition of 100 μl of 10 mM Tris Base into each well.Plates were gently shaken and left at room temperature for 30 minutesbefore measuring the optical density at 564 nM on a Microquantmicrotiter plate reader.

The results are detailed below in table 2. LY294002 exhibited anEnhancement Ration of 1.09.

PI 3-Kinase Inhibition

In order to assess the inhibitory action of the compounds against PI3-kinase in vitro, the following assay was used to determine IC₅₀values.

Baculoviral recombinant GST-fused PI 3-kinase (p110α/p85α) was purifiedfrom Sf9 insect cells using GSH-sepharose affinity chromatography asdescribed (Wymann, M. T et al., (1996) Wortmannin inactivatesphosphoinositide 3-kinase by covalent modification of Lys-802, a residueinvolved in the phosphate transfer reaction. Mol. Cell Biol.16:1722-1733). PI 3-kinase (1 μl) was diluted in reaction buffer (89 μlof 50 mM Hepes pH 7.5, 150 mM NaCl, 0.1 mM Sodium Orthovanadate,containing 20 μg of phosphatidylinositol) and varying concentrations ofinhibitor compound added. All compounds were diluted in DMSO to give afinal assay concentration of between 100 and 0.1 μM, with DMSO being ata final concentration of 1%. After 10 minutes of incubation at 37° C.the reactions were initiated by the addition of 10 μl of 50 μM Na-ATP,20 mM MgCl₂ and 2.5 μCi ³³p-γATP. Reactions were incubated for a further20 minutes at 37° C., before quenching with the addition of 400 μl ofchloroform/methanol (1:1). Reactions were acidified by the addition of200 μl of 1M HCl, before separation of the organic and aqueous phases bycentrifugation at 10,000 g for 30 seconds. The organic phase wastransferred to a fresh tube and washed twice with 150 μl of 1Mhydrochloric acid/methanol (1:1), discarding the aqueous phase. Thewashed reaction product was then placed in a white 96-well plate with100 μl of scintillation fluid and transferred to a TopCount NXT forscintillation counting. Counts per minute, following a one minutecounting time, were recorded for each reaction. The inhibition of PI3-kinase activity by compounds was calculated as described above for theDNA-PK assay.

The selectivity was determined by the following equation:

${\Delta\left( {{DNA} - {{{PK}/{PI}}\; 3} - K} \right)} = \frac{{IC}_{50}\left( {{{PI}\; 3} - K} \right)}{{IC}_{50}\left( {{DNA} - {PK}} \right)}$

The results are detailed below in table 3. 294 exhibited an IC₅₀ of 1.5μM, and a Δ(DNA-PK/PI 3-K) of 1.

ATM inhibition

In order to assess the inhibitory action of the compounds against ATM invitro, the following assay was used to determine IC₅₀ values.

ATM protein was immunoprecipitated from HeLa cell nuclear extract usingrabbit polyclonal antisera raised to the C-terminal ˜500 amino-acidresidues of the human ATM protein. The immunoprecipitation was performedaccording to the methodology described by Banin, S. et al. (1998)Enhanced phosphorylation of p53 by ATM in response to DNA damage.Science 281:1674-1677. 10 μl of immunoprecipitated ATM in Buffer C (50mM Hepes, pH 7.4, 6 mM MgCl2, 150 mM NaCl, 0.1 mM sodium orthovanadate,4 mM MnCl2, 0.1 mM dithiothreitol, 10% glycerol) was added to 32.5 μl ofbuffer C containing 1 μg of the ATM substrate GSTp53N66 in a V-bottomed96 well polypropylene plate. The GSTp53N66 substrate is the aminoterminal 66 amino acid residues of human wild type p53 fused toglutathione S-transferase. ATM phosphorylates p53 on the residue serine15 (Banin, S. et al. (1998) Enhanced phosphorylation of p53 by ATM inresponse to DNA damage. Science 281:1674-1677). Varying concentrationsof inhibitor were then added. All compounds were diluted in DMSO to givea final assay concentration of between 100 and 1 μM, with DMSO being ata final concentration of 1%. After 10 minutes of incubation at 37° C.,the reactions were initiated by the addition of 5 μl of 50 μM Na-ATP.After 1 h with shaking at 37° C., 150 μl of phosphate buffered saline(PBS) was added to the reaction and the plate centrifuged at 1500 rpmfor 10 minutes. 5 μl of the reaction was then transferred to a 96 wellopaque white plate containing 45 μl of PBS to allow the GSTp53N66substrate to bind to the plate wells. The plate was covered andincubated at room temperature for 1 h with shaking before discarding thecontents. The plate wells were washed twice by the addition of PBS priorto the addition of 3% (w/v) bovine serum albumin (BSA) in PBS. The platewas incubated at room temperature for 1 h with shaking before discardingthe contents and washing twice with PBS. To the wells, 50 μl of a1:10,000 dilution of primary phosphoserine-15 antibody (Cell SignalingTechnology, #9284L) in 3% BSA/PBS was added to detect thephosphorylation event on the serine 15 residue of p53 elicited by theATM kinase. After 1 h of incubation at room temperature with shaking,the wells were washed four times with PBS prior to the addition of ananti-rabbit HRP conjugated secondary antibody (Pierce, 31462) withshaking for 1 h at room temperature. The wells were then washed fourtimes with PBS before the addition of chemiluminescence reagent (NENRenaissance, NEL105). The plate was then shaken briefly, covered with atransparent plate seal and transferred to a TopCount NXT forchemiluminescent counting. Counts per second, following a one secondcounting time, were recorded for each reaction. The inhibition of ATMactivity by compounds was calculated as described above for the DNA-PKassay.

The selectivity was determined by the following equation:

${\Delta\left( {{DNA} - {{PK}/{ATM}}} \right)} = \frac{{IC}_{50}({ATM})}{{IC}_{50}\left( {{DNA} - {PK}} \right)}$

The results are detailed below in table 4. 294 exhibited an IC₅₀ of >100μM, and a Δ(DNA-PK/ATM) of >67.

All the compounds showed activity in DNA-PK inhibition, exhibiting anIC₅₀ of less than about 12 μM and/or % inhibition at 1 μM of more thanabout 22%.

Selected compounds and their IC₅₀ values are listed in table 1.

Compounds which exhibited particular efficacy in DNA-PK inhibition,having an IC₅₀ of less than about 1 μM and/or % inhibition of more thanabout 50 at 1 μM, include 270, 271, 272, 279, 267, 269, 268, 59, 60, 73,131, 123, 139, 74, 125, 126, 127, 99, 124, 140, 143, 118, 105, 106, 104,146, 107, 114, 163, 215, 194, 166, 187, 167, 157, 200, 169, 170, 202,211, 173, 175, 176, 178, 179, 190, 192, 212, 182, 214, 203, 198, 205,206, 264, 242, 258, 260, 247, 249, 252, 253, 255, 37, 31, 64, 65, 32,68, 35, 36, 72, 293, 301, 297, 283, 287, 289, 288, 304, 5, 1, 292, 291,290, 3, 4, 337, 418, 416, 422, 415, 6, 318, 338, 339, 340, 341, 426,317, 366, 375, 385, 403, 404, 408, 409, 410, 389, 394 and 413.

TABLE 1 DNA-PK Inhibition Compound Number IC₅₀ (μM) 1 1.0 3 0.5 4 0.45 22.5 5 0.35 12 10.5 13 8 6 5 285 0.8 284 0.35 287 0.4 289 0.45 286 0.3288 0.35 292 0.25 291 0.3 290 0.4 304 0.8 307 0.9 425 2.0 337 0.65 4233.0 418 0.45 414 1.5 416 0.6 419 1.0 422 0.5 415 0.5 343 0.7 338 0.95341 0.65 342 0.8 293 0.4 301 0.4 297 0.5 296 1.2 312 10 310 0.1 330 20317 0.3

TABLE 2 Enhancement Ratio Compound Number ER 3 1.5 4 2.0 5 1.63 285 1.62284 1.72 287 1.61 289 1.87 286 1.5 288 1.69 292 1.16 291 1.26 337 1.12414 1.69 416 1.32 422 1.68 415 1.86 293 1.7 297 2.12 310 1.73 317 3.62

TABLE 3 PI 3-kinase Inhibition Compound Number IC₅₀ (μM) Δ(DNA-PK/PI3-K) 3 10 20 4 7 16 5 750 >143 285 30 38 283 10 29 289 37 82 288 15 43292 13 52 414 95 63 422 6 12 415 >100 >200 293 20 50 301 16 40 297 18 36296 6 5 310 11 110 317 2.5 8

TABLE 4 ATM Inhibition Compound Number IC₅₀ (μM) Δ(DNA-PK/ATM)3 >50 >100 4 >100 >222 2 >50 >20 5 >100 >286 285 >50 >63 284 >100 >286287 >50 >125 289 >50 >111 286 24 80 288 >100 >286 292 >100 >400291 >50 >167 290 35 88 304 >100 >125 307 >100 >111 337 >100 >154423 >100 >33 418 >100 >222 414 >100 >67 416 >100 >167 422 >100 >200415 >100 >200 293 >100 >250 301 >50 >125 297 45 90 296 >100 >83310 >100 >1000 317 >100 >333

1. A method for the treatment of tumours comprising administering to asubject suffering from tumour growth a therapeutically-effective amountof a compound of formula Ia or Ib:

and isomers, salts, and solvates, thereof, in combination with ionisingradiation or one or more chemotherapeutic agents, wherein: R¹ and R² areindependently hydrogen, an optionally substituted C₁₋₇ alkyl group,C₃₋₂₀ heterocyclyl group, or C₅₋₂₀ aryl group, or may together form,along with the nitrogen atom to which they are attached, an optionallysubstituted heterocyclic ring having from 4 to 8 ring atoms; and R³ isan optionally substituted C₅₋₂₀ aryl group.
 2. The method according toclaim 1, wherein R¹ and R² together with the nitrogen atom to which theyare attached form a morpholino group.
 3. The method according to claim1, wherein R³ is optionally substituted phenyl.
 4. The method accordingto claim 3, wherein R³ is phenyl substituted with a substituent selectedfrom halo, C₁₋₇ alkyl, ether, alkoxy, nitro, cyano, acyl, formyl, ester,acyloxy, hydroxy, carboxy, C₅₋₂₀ aryl, C₃₋₂₀ heterocyclyl, acylamido,acylureido, thioureido, carbamate, carbazoyl, amido and amino.
 5. Themethod according to claim 3, wherein R³ is a mono-substituted phenyl. 6.The method according to claim 5, wherein R³ is a 4-substituted phenyl.7. The method according to claim 3, wherein the phenyl substituent isfurther substituted by halo, nitro, cyano, hydroxy, ester, ether,acyloxy, acyloxy, acyl, thioether, carboxy, amino C₅₋₂₀ aryl, C₁₋₇ alkylor C₃₋₂₀ heterocyclyl.